Inhibitors of beta integrin-G protein alpha subunit binding interactions

ABSTRACT

Provided herein are compounds that inhibit a binding interaction between a β integrin and a G protein subunit, as well as compositions, e.g., pharmaceutical compositions, comprising the same, and related kits. In some embodiments, the compound is an antibody or antibody analog, and, in other embodiments, the compound is a peptide or peptide analog. Also provided are methods of using the compounds, including methods of treating or preventing a medical condition, such as stroke, heart attack, cancer, or inflammation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/843,152, filed Sep. 2, 2015, now U.S. Pat. No. 10,011,634, which is adivisional of U.S. application Ser. No. 14/176,930, filed Feb. 10, 2014,now U.S. Pat. No. 9,156,884, which is a continuation of U.S. applicationSer. No. 13/621,064, filed on Sep. 15, 2012, now U.S. Pat. No.8,685,921, which claims priority to International Patent Application No.PCT/US2011/028567, filed Mar. 15, 2011, which claims priority to U.S.Provisional Patent Application No. 61/314,027, filed on Mar. 15, 2010,and U.S. Provisional Patent Application No. 61/433,037, filed on Jan.14, 2011; each application of which is incorporated by reference intheir entirety.

GRANT FUNDING

This invention was made with government support under Grant Nos.HL080264, HL062350, HL068819, awarded by the National Heart, Lung, andBlood Institute. The government has certain rights in the invention.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: 89 kilobytes ACII (Text) file named“45326B_SeqListing,” created on Sep. 14, 2012.

BACKGROUND

Integrins are heterodimer transmembrane receptors for the extracellularmatrix and are composed of an alpha and beta subunit.Naturally-occurring integrin ligands include laminin, fibronectin, andvitronectin, and also include fibrinogen and fibrin, thrombospondin, andvon Willebrand factor, and fibroblast growth factor 2. Integrins bindligands by recognizing short amino acid stretches on exposed loops,particularly the arginine-glycine-aspartic acid (RGD) or like sequences.Upon ligation, integrins mediate cell adhesion, and initiate complexsignaling events, alone or in combination with other types of receptors(such as growth factor receptors), and regulate cell spreading,retraction, adhesion, proliferation, survival, and migration. Integrinsignaling is bi-directional. Intracellular signals mediates so-called“inside-out” signaling, which induces activation of the ligand bindingfunctions of integrins. Integrin ligation activate “outside-in”signalingpathways, including, for example Src family kinases (SFK), thephosphoinositide 3-kinase, protein kinase B (PKB/Akt), mitogen-activatedprotein kinase (MAPK), and Rac. See, e.g., Li Z, Delaney M K, O'Brien KA, Du X., Arterioscler Thromb Vase Biol. 30(12):2341-2349, 2010.

Integrins are expressed and serve as major adhesion receptors on thesurface of blood platelets, a type of blood cells that are critical inthrombosis and hemostasis. The major integrin expressed on plateletsurface is the integrin αIIbβ3, also called glycoprotein IIb-IIIa(GPIIa-IIIa). Upon exposure to the site of vascular injury, plateletsadhere to and spread on the injured or stimulated vascular endothelialcells or extracellular matrix, becomes activated and aggregate to fromprimary thrombus. Integrin αIIbβ3 mediates stable platelet adhesion,spreading and aggregation. This process normally serves to stop bleedingand prevent loss of blood (that is called hemostasis). Under certainconditions, such as at sites of atherosclerosis, platelets form aocclusive thrombus that block blood vessels, leading to ischemia oforgans and tissues, causing such as heart attack and thrombotic strokeetc (Li Z, Delaney M K, O'Brien K A, Du X., Arterioscler Thromb VaseBiol. 30(12):2341-2349, 2010.). Thus, inhibitors of integrin functionare clinically used to prevent and treat thrombotic diseases. Integrinsare also important in other physiological and pathological processessuch as immunity, inflammation, angiogenesis and tumor progression andmetastasis.

Three classes of integrin inhibitors are currently in clinical use ordevelopment: monoclonal antibodies targeting the extracellular ligandbinding domain of the heterodimer (eg, Reopro, Eli Lilly, Indiapolis,Vitaxin; MedImmune, Gaithersburg, Md.), synthetic peptides containing anRGD or KGD sequences (eg, Integrillin, Millennium Pharmaceuticals;cilengitide; Merck KGaA, Darmstadt, Germany), and peptidomimetics (eg,aggrestat (Tirofiban), Merck, White House Station, N.J.; S247; Pfizer,St Louis, Mo.).

The first integrin-specific drugs targeted the integrin α_(IIb)β₃, whichis central to hemostasis and plays an important role in plateletadhesion and thrombus formation. α_(IIb)β₃ also functions in theinflammatory response. The first FDA-approved α_(IIb) β₃ antagonistshave proven benefit for indications, including acute coronary syndromesand prevention of myocardial infarction. However, the use of some ofthese drugs are limited due to their pharmacokinetic profiles—some drugsdemonstrate rapid plasma clearance, rapid metabolism, poor oralbioavailability, and/or large variation in plasma levels. Also, someantagonists of α_(IIb)β₃ integrin induced thrombocytopenia. See, e.g.,Advances in Immunology, Volume 91, Elsevier Academic Press (San Diego,Calif.), 2006. A common and potentially life-threatening adverse effectof integrin inhibitor is bleeding (this is because intergrin is impotantin hemosrsis).

SUMMARY

The invention provides a compound that inhibits a binding interactionbetween a β integrin and a G protein a subunit. As further discussedherein, in exemplary embodiments, the compound takes form of anantibody, or antibody analog, a peptide, or peptide analog. Accordingly,the invention provides, antibodies, antibody analogs, peptides, andpeptide analogs.

Also provided by the invention is a composition, e.g., a pharmaceuticalcomposition, comprising a compound that inhibits a binding interactionbetween a β integrin and a G protein a subunit. Kits comprising one ormore compounds of the are also provided herein.

Methods of using the compounds and compositions of the invention arefurther provided. For example, the invention provides a method ofinhibiting a binding interaction between an integrin and a G proteinsubunit in a cell. The method comprises the step of contacting the cellwith a compound or composition of the invention in an amount effectiveto inhibit the binding interaction.

The invention further provides a method of inhibiting integrin-dependentSrc activation in a cell. The method comprises the step of contactingthe cells with a compound or composition of the invention in an amounteffective to inhibiting the Src activation.

A method of activating a GTPase is furthermore provided by theinvention. The method comprises the step of contacting a G proteinsubunit with a compound or composition in an amount effective toactivate a GTPase.

The invention moreover provides a method of inhibiting spreading ormigration of a cell. The method comprises the step of contacting thecell with a compound or composition of the invention in an amounteffective to inhibit spreading and migration.

Also provided by the invention is a method of inhibiting plateletadhesion. The method comprises the step of contacting a platelet with acompound or composition of the invention in an amount effective toinhibit platelet adhesion. The invention further provides a method ofinhibiting platelet granule secretion and platelet aggregation. Themethod comprises the step of contacting a platelet with a compound orcomposition of the invention in an amount effective to inhibit granulesecretion and aggregation.

The compounds and compositions of the invention are additionallycontemplated for therapeutic purposes. For example, the compounds andcompositions of the invention may be used to enhance blood clotretraction or inhibit thrombosis in a subject in need thereof.Accordingly, the invention provides a method of enhancing blood clotretraction in a subject in need thereof. The method comprises the stepof administering to the subject a compound or composition of theinvention in an amount effective to enhance blood clot retraction. Alsoprovided is a method of inhibiting thrombosis in a subject in needthereof. The method comprises the step of administering to the subject acompound or composition of the invention in an amount effective toinhibit thrombosis.

Because thrombosis play a role in stroke and heart attack, the inventionfurthermore provides a method of treating or preventing a stroke or aheart attack in a subject in need thereof. The method comprises the stepof administering to the subject a compound or composition of theinvention in an amount effective to treat or prevent stroke or heartattack.

Because the compounds and compositions provided herein relate to thecoordinated cell spreading-retraction process, which in turn, isimportant in cell migration, the invention also provides a method ofinhibiting metastasis of a tumor cell. The method comprises the step ofcontacting a tumor cell with a compound or composition of the inventionin an amount effective to inhibit metastasis. The compounds andcompositions are also contemplated for use in inhibiting angiogenesis.Accordingly, the invention provides a method of inhibiting angiogenesisin a subject in need thereof. The method comprises the step ofadministering to the subject a compound or composition of the inventionin an amount effective to inhibit angiogenesis.

Since metastasis and angiogenesis are important aspects of cancer, theinvention moreover provides a method of treating or preventing cancer ina subject in need thereof. The method comprises the step ofadministering to the subject a compound or composition of the inventionin an amount effective to treat or prevent cancer.

The compounds and compositions provided herein also may be used foraffecting leukocyte function. The invention accordingly provides amethod of inhibiting leukocyte adhesion, spreading, migration, orchemotaxis. The method comprises the step of contacting a leukocyte witha compound or composition of the invention in an amount effective toinhibit leukocyte adhesion, spreading, migration, or chemotaxis. Sincethese leukocyte functions are related to inflammation, the inventionadditionally provides a method of inhibiting or treating inflammation ina subject in need thereof. The method comprises the step ofadministering to the subject a compound or composition of the inventionin an amount effective to inhibit or treat inflammation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E demonstrate a role of Gα₁₃ in integrin outside-in signaling.(FIG. 1A) Confocal microscopy images of spreading scrambled siRNAcontrol platelets or Gα₁₃-depleted platelets (Gα₁₃-siRNA) on fibrinogen,without or with Y27632. Merged EGFP (green) fluorescence and Alex Fluor546-conjugated phalloidin (Red) fluorescence. (FIG. 1B) Western blotcomparison of Gα₁₃ abundance in platelets from mice innoculated withcontrol siRNA- or Gα₁₃-siRNA-transfected bone marrow stem cells. (FIG.1C, FIG. 1D, FIG. 1E) Mouse platelets from scrambled siRNA- or Gα₁₃siRNA-transfected stem cells were allowed to adhere to immobilizedfibrinogen, solubilized and analyzed for c-Src Tyr⁴¹⁶ phosphorylationand RhoA activation.

FIGS. 2A-2G demonstrate the binding of Gα₁₃ to β₃ and the inhibitoryeffect of mSRI peptide. (FIG. 2A) Proteins from platelet lysates wereimmunoprecipitated with control IgG or antibody to Gα₁₃ with or without1 μM GDP, 1 μM GTP or 30 μM AlF₄ ⁻. Immunoprecipitates wereimmunoblotted with anti-Gα₁₃ or anti-β₃ (MAb15). See FIG. S4 forquantitation. (FIG. 2B) Proteins from platelet lysates wereimmunoprecipitated with control mouse IgG, anti-α_(IIb)β₃ (D57 (24)) oran antibody to the glycoprotein Ibα (GPIb). Immunoprecipitates wereimmunoblotted with anti-Gα₁₃, anti-β₃, or anti-GPIb antibodies. (FIG.2C, FIG. 2D) Purified GST-β3CD (FIG. 2C) or GST-β1CD (FIG. 2D) bound toglutathione beads was mixed with purified Gα₁₃ with or without 1 μM GDP,1 μM GTPγS or 30 μM AlF₄ ⁻. Bound proteins were immunoblotted withanti-Gα₁₃. Quantitative data are shown as mean±SD and p value (t-test).(FIG. 2E) Lysates of control platelets or platelets adherent tofibrinogen in the absence or presence of 0.025 U/ml thrombin wereimmunoprecipitated with anti-Gα₁₃, and then immunoblotted with MAb15.Quantitative data are shown as mean±SD and p value (t-test). (FIG. 2F)Lysates from 293FT cells transfected with Flag-tagged wild type Gα₁₃ orindicated truncation mutants (see FIG. S5) were precipitated withGST-β₃CD- or GST-bound glutathione beads. Bead-bound proteins wereimmunoblotted with anti-Flag (Bound). Flag-tagged protein amounts inlysates are shown by anti-Flag immunoblot (Input). (FIG. 2G) Proteinfrom platelet lysates treated with 0.1% DMSO, 250 μM scrambled controlpeptide (Ctrl) or mSRI were immunoprecipitated with anti-Gα₁₃.Immunoprecipitates were immunoblotted with anti-Gα₁₃ or anti-β₃. SeeFIG. S4 for quantitation.

FIGS. 3A-3B demonstrate the effects of mSRI on integrin-induced c-Srcphosphorylation, RhoA activity and platelet spreading. (FIG. 3A) Washedhuman platelets pre-treated with DMSO, mSRI, or scrambled controlpeptide were allowed to adhere to fibrinogen and then solubilized atindicated time points. Proteins from lysates were immunoblotted withantibodies to c-Src phosphorylated at Tyr⁴¹⁶, c-Src, or RhoA. GTP-boundRhoA was measured by association with GST-RBD beads (25). See FIG. S4for quantitative data. (FIG. 3B) Spreading of platelets treated with0.1% DMSO, scrambled control peptide, or mSRI, in the absence orpresence of C3 toxin, Y27632, or 0.01 U/ml thrombin. Platelets werestained with Alexa Fluor 546-conjugated phalloidin.

FIGS. 4A-4G demonstrate a role of Gα₁₃ in clot retraction and dynamicRhoA regulation. (FIG. 4A) Effect of 250 μM mSRI peptide on clotretraction of human platelet-rich plasma compared with DMSO andscrambled peptide. Clot sizes were quantified using Image J (mean±SD,n=3, t-test). (FIG. 4B) Comparison of clot retraction (mean±SD, n=3,t-test) mediated by control siRNA platelets and Gα₁₃-depleted platelets.(FIG. 4C, FIG. 4D, FIG. 4E, FIG. 4F) Platelets were stimulated withthrombin with or without 2 mM RGDS, and monitored for turbidity changesof platelet suspension caused by shape change and aggregation (FIG. 4C).The platelets were then solubilized at indicated time points andanalyzed for amount of β₃ coimmunoprecipitated with Gα₁₃ (FIG. 4D) andamount of GTP-RhoA bound to GST-RBD beads (FIG. 4E) by immunoblot. (FIG.4F) quantitative data (mean±SD) from 3 experiments. (FIG. 4G) Aschematic illustrating Gα₁₃-dependent dynamic regulation of RhoA andcrosstalk between GPCR and integrin signaling.

FIG. 5 demonstrates the efficiency of platelet replacement byirradiation and transplantation of lentivirus-infected bone marrow stemcells. Five weeks after high dose irradiation and transplantation ofbone marrow stem cells infected with scrambled siRNA- or Gα₁₃-specificsiRNA #1-encoding lentivirus, platelets were isolated from recipientmice, and allowed to adhere to immobilized fibrinogen. Platelets wereimaged using a Zeiss LSM 510 META confocal microscope. Green: EGFPfluorescence indicating that platelets are derived from transplantedstem cells. Red: Alexa Fluor 546-conjugated phalloidin stainingindicating total platelets.

FIGS. 6A-6C demonstrate the similar effects of two different Gα13 siRNAon platelet spreading, c-Src activation and RhoA activity, and theeffect of aspirin on platelet spreading. (FIG. 6A) Confocal microscopyimages of spreading mouse platelets transfected with scrambled siRNA,Gα₁₃ siRNA #1- and Gα₁₃ siRNA #2 on immobilized fibrinogen. Merged EGFPgreen fluorescence and Alexa Fluor 546-conjugated phalloidin redfluorescence. (FIG. 6B) Scrambled siRNA, Gα₁₃ siRNA #1- and Gα₁₃ siRNA#2-transfected platelets were allowed to adhere to immobilizedfibrinogen for the indicated time, and analyzed for c-Src activation andRhoA activity. Note that two different siRNA similarly inhibitedplatelet spreading and c-Src activation, and accelerated activation ofRhoA. (FIG. 6C) Mouse platelets were pre-incubated with or without 1 mMaspirin for 30 minutes at room temperature, and then allowed to spreadon immobilized fibrinogen.

FIGS. 7A-7C demonstrate the inhibitory effects of Gα₁₃ siRNA on cellspreading and c-Src phosphorylation in CHO cells expressing integrinα_(IIb)β₃ and its rescue by an siRNA resistant mutant of Gα₁₃. StableCHO cell line expressing integrin α_(IIb)β₃ (123 cells) were transfectedwith cDNA constructs encoding EGFP and scrambled control siRNA or Gα₁₃siRNA with or without co-transfection of Flag-tagged siRNA-resistantmutants of Gα₁₃ cDNA constructs (Flag-G13-Mut1). (FIG. 7A) Cells weresolubilized and immunoblotted with anti-Gα₁₃ antibody, anti-Flag (fordetecting Flag-tagged Gα₁₃) and an antibody to tubulin (loadingcontrol). (FIG. 7B) Cells were plated on fibrinogen-coated surfaces forvarious lengths of time, solubilized, and lysates were immunoblotted forc-Src phosphorylation at Y416 to indicate c-Src activation, or totalamount of c-Src. (FIG. 7C) Cells adherent to fibrinogen were stainedwith Alexa Fluor 633-labeled phalloidin (artificial blue color) andAlexa Fluor 546-conjugated anti-Flag antibody (Red), and imaged using aZeiss LSM 510 META confocal microscope. Cells that were successfullytransfected with siRNA constructs express EGFP and thus show greenfluorescence. Note that Gα₁₃ siRNA inhibited integrin-dependent c-Srcactivation and cell spreading on fibrinogen, which was rescued byexpressing Flag-G13-Mut1.

FIGS. 8A-8D provide quantitative data from experiments shown in FIGS.2A-2G and 3A-3B. (FIG. 8A) Quantitative data for FIG. 2A, showing thatco-immunoprecipitation of β₃ with Gα₁₃ was enhanced by GTP and AlF4⁻.(FIG. 8B) Quantitative data for FIG. 2G, showing mSRI inhibitedco-immunoprecipitation of β₃ with Gα₁₃. (FIG. 8C and FIG. 8D)Quantitative data from FIG. 3A showing that mSRI inhibited c-Srcactivation (FIG. 8C) and accelerated RhoA activation (FIG. 8D). All dataare expressed as mean±SD from 3 experiments. Statistical significancewas determined using Student t-test.

FIG. 9 represents a schematic of Gα13 with switch regions indicated. TwoGα₁₃ truncation mutants were developed to map the β₃ binding site (SeeFIG. 2F): (1) the mutant encoding a Gα₁₃ fragment containing residues1-196 lacking switch region I, and (2) the mutant encoding a Gα₁₃fragment (residues 1-212) containing switch region I.

FIGS. 10A-10B provide typical images of clot retraction showing theeffects of mSRI and Gα₁₃-knockdown on platelet-mediated clot retraction.(FIG. 10A) Effect of 250 μM mSRI peptide on clot retraction of humanplatelet-rich plasma compared with DMSO and scrambled peptide.Quantitative data are shown in FIG. 4A. (FIG. 10B) Comparison of clotretraction mediated by control siRNA platelets and Gα₁₃-knockdownplatelets. Quantitative data are shown in FIG. 4B.

FIGS. 11A-11E demonstrate the Gα₁₃ binding site in the integrin β₃cytoplasmic domain. (FIG. 11A) Amino acid sequence alignment (SEQ IDNOs: 12-18, respectively) of the cytoplasmic domains of various humanintegrins β subunits (Inhibitor peptides—SEQ ID NOs: 21, 22 and 83,respectively). Key sequences critical in Gα13 binding is marked as red.Synthetic peptides corresponding to the Gα13 binding region of β3 weresynthesized (FIG. 11B) Lysates from CHO cells expressing a similar levelof wild type and truncated integrin β₃ were immunoprecipitated withanti-Gα₁₃ antibody or equal amount of control rabbit IgG. Lysates andimmunoprecipitates were immunoblotted with anti-Gα₁₃ or anti-β₃ (MAb15)antibody. (FIG. 11C) Lysates from CHO cells expressing wild type ormutant β₃ were immunoprecipitated with anti-Gα₁₃ antibody or equalamount of control rabbit IgG. Immunoprecipitates were immunoblotted withanti-Gα₁₃ or anti-β₃ (MAb 15) monoclonal antibodies. (FIG. 11D) Confocalmicroscopy images of β₃ ^(−/−) platelets, and β₃ ^(−/−) plateletsexpressing wild type or AAA-mutant integrin β₃ spreading on fibrinogen.Merged anti-integrin β₃ (MAb15) fluorescence (green) and Alexa Fluor546-conjugated phalloidin fluorescence (red). (FIG. 11E) Flow cytometryanalysis of β₃ expression levels in β₃ ^(−/−) mouse plateletstransfected with wild type and AAA (E⁷³¹⁻⁷³³ to alanine) mutant integrinβ₃ in complex with wild type α_(IIb). β₃ ^(−/−) platelets serve asnegative control. (b)

In FIG. 12A, CHO cells expressing wild type or AAA mutant α_(IIb)β₃ wereallowed to spread on fibrinogen surfaces. (FIG. 12B) Wild-type orAAA-mutant α_(IIb)β₃-expressing CHO-1b9 cells were allowed to adhere toimmobilized fibrinogen, solubilized and analyzed for RhoA activation andc-Src Tyr⁴¹⁶ phosphorylation. (FIG. 12C) Quantitation of RhoA activityand c-Src Tyr⁴¹⁶ phosphorylation as shown in (d) (mean±SD, 3experiments). (FIG. 12D) Flow cytometry analysis of PAR4AP-inducedOregon-green-labeled fibrinogen binding to wild-type or AAA-mutantα_(IIb)β₃-expressing platelets from bone marrow-transplanted β₃ ^(−/−)mice. β₃ ^(−/−) platelets served as negative control.

FIGS. 13A-13E demonstrate the identification of a Gα₁₃ binding motif inintegrins. (FIG. 13A) Platelets were treated with 500 μM controlpeptide, mP₁₃, mP₇, or mP₅ peptides, then immunoprecipitated withanti-Gα₁₃ antibody or equal amount of control rabbit IgG. Lysates andimmunoprecipitates were immunoblotted with anti-Gα₁₃ or anti-β₃ (MAb15)antibody. (FIG. 13B) Human platelets treated with DMSO, Myr-Scrambledpeptide or mP₅ peptide were allowed to adhere to immobilized fibrinogen,solubilized and analyzed for RhoA activation and c-Src Tyr⁴¹⁶phosphorylation. (FIG. 13C) Confocal microscopy images of plateletstreated with DMSO, Myristoylated Scrambled peptides, and mP₅ peptidetreated platelets adherent on fibrinogen for 30 minutes, without or withRho kinase inhibitor Y27632. Merged integrin 33 (green) fluorescence andAlexa Fluor 546-conjugated phalloidin (red) fluorescence. (FIG. 13D)Flow cytometry analysis of PAR4AP (50 μM)-induced Oregon-green labelledfibrinogen binding to human platelets pre-treated with DMSO, 250 μMMyr-Scrambled peptide or 250 μM mP₅ peptide. (FIG. 13E) Platelets werepreincubated with mP5 or scrambled control peptides and then stimulatedwith thrombin in a platelet aggregometer at 37° C. Platelet aggregationtraces were recorded.

FIGS. 14A-14B demonstrate the inhibition of integrin outside-in andinside-out signalling by mP₁₃ peptide. (FIG. 14A) Fluorescencemicroscopy images of human platelets adherent on fibrinogen that werepretreated with 250 μM myristoylated scrambled control peptide, 250 μMmP₁₃ peptide. (FIG. 14B) Flow cytometry analysis of PAR4AP (50μM)-induced Oregon-green labelled fibrinogen binding to human plateletspre-treated with DMSO, 150 μM Myr-Scrambled peptide or 15 μM mP₁₃peptide.

FIGS. 15A-15E demonstrate that the EEE motif of integrin β3-CD isimportant for Gα13 binding and cell spreading. (FIG. 15A) Human integrincytoplasmic domain sequences were aligned manually (SEQ ID NOs: 12-15,respectively; Inhibitor peptides—SEQ ID NOs: 21, 22 and 83,respectively). The conserved ExE motifs are highlighted in red. Theconserved NxxY and HDR[R/K] motifs are highlighted in bold. Residues arenumbered according to the National Center for Biotechnology Information(NCBI) sequence. The sequences of the peptide inhibitors developed inthis study are shown below the corresponding β3 cytoplasmic domainsequences. (FIG. 15B) Lysates from truncated integrin β3-stableexpression 123 cells were precipitated with anti-Gα13 antibody or equalamount of normal rabbit IgG as control. Immunoprecipitates wereimmunoblotted with anti-Gα13 or anti-β3 (M15) antibody. (FIG. 15C)Lysates from E to A mutant integrin β3-stable expression 123 cells wereprecipitated with anti-Gα13 antibody or equal amount of normal rabbitIgG as control. Immunoprecipitates were immunoblotted with anti-Gα13 oranti-β3 (M15) antibody. (FIG. 15D) Confocal microscopy images ofspreading β3−/− control platelets or platelets express wide type orAAA-mutant integrin β3 on fibrinogen. Merged integrin β3 (green)fluorescence and Alex Fluor 546-conjugated phalloidin (Red)fluorescence. (FIG. 15E) Flow cytometry analysis of wide type andAAA-mutant integrin β3 expression level. Human platelets and β3−/−platelets serve as positive and negative control.

FIGS. 16A-16H demonstrate that the AAA mutation is responsible forincreased RhoA activity and decreased c-Src activity without affectingintegrin inside-out signaling. (FIG. 16A) Wide type or AAA-mutant123cells were allowed to adhere to immobilized fibrinogen, solubilized andanalyzed for RhoA activation and c-Src Tyr416 phosphorylation. (FIG.16B) Quantitation of RhoA activity for FIG. 16A. (FIG. 16C) Quantitationof c-Src Tyr416 phosphorylation for FIG. 16A. (FIG. 16D) Lysates fromwide type or AAA-mutant integrin β3-stable expression 123 cells wereprecipitated with anti-c-Src antibody or equal amount of normal rabbitIgG as control. Immunoprecipitates were immunoblotted with anti-v-Src oranti-p190RhoGAP antibody. (FIG. 16E) Quantitation of c-Src boundp190RhoGAP for FIG. 16D. (FIG. 16F) Lysates from wide type or AAA-mutantintegrin β3-stable expression 123 cells were precipitated withanti-p115RhoGEF antibody or equal amount of normal mouse IgG as control.Immunoprecipitates were immunoblotted with anti-Gα13 or anti-p115RhoGEFantibody. (FIG. 16G) Quantitation of p115RhoGEF bound Gα13 for FIG. 16F.(FIG. 16H) Flow cytometry analysis of wide type and AAA-mutant integrinβ3-expression platelets fibrinogen binding ability induced by Par4-.Human platelets and β3−/− platelets serve as positive and negativecontrol.

FIGS. 17A-17I demonstrate that the Myr-P5 peptide inhibited plateletsaggregation and spreading due to increased RhoA activity and decreasedc-Src activity without affecting integrin inside-out signaling. (FIG.17A) Human platelets treated with DMSO, Myr-Scramble or Myr-P5 peptidewere allowed to adhere to immobilized fibrinogen, solubilized andanalyzed for RhoA activation and c-Src Tyr416 phosphorylation. (FIG.17B) Quantitation of RhoA activity for FIG. 17A. (FIG. 17C) Quantitationof c-Src Tyr416 phosphorylation for FIG. 17A. (FIG. 17D) Lysates formDMSO, Myr-Scramble or Myr-P5 peptide treated platelets were precipitatedwith anti-Gα13 antibody or equal amount of normal rabbit IgG as control.Immunoprecipitates were immunoblotted with anti-Gα13 or anti-β3 (M15)antibody. (FIG. 17E) Quantitation of Gα13-bound integrin β3 for FIG.17D. (FIG. 17F) Confocal microscopy images of DMSO, Myr-Scramble orMyr-P5 peptide treated platelets spreading on fibrinogen for 30 minutes,without or with Y27632. Merged integrin β3 (green) fluorescence and AlexFluor 546-conjugated phalloidin (Red) fluorescence. (FIG. 17G, FIG. 17H)Flow cytometry analysis of DMSO, Myr-Scramble or Myr-P5 peptide treatedplatelets fibrinogen binding ability induced by Par4. Peptideconcentration was 150 μM for FIG. 17G, 250 μM for H. (FIG. 17I) 200 μMMyr-P5 peptide inhibited platelets aggregation induced by thrombin.

FIGS. 18A-18F demonstrate that Talin head could not rescue AAA-mutant123 cells spreading-deficiency on fibrinogen. (FIG. 18A, FIG. 18C)Purified GST-β3-CD bound to glutathione beads was mixed with purifiedGα13 with or without purified talin. Bound proteins were immunoblottedwith anti-Gα13 or anti-talin antibody. Quantitative data are shown asmean±SD and p value (ttest). (FIG. 18B, FIG. 18D) Quantitation ofGST-β3-CD bound Gα13 or talin for FIG. 18A and FIG. 18C. (FIG. 18E)Lysates from 123 or AAA cells transfected with talin head wereprecipitated with anti-β3 rabbit serum (8053) or eqival amount ofpre-immune serum. Immunoprecipitates were immunoblotted with anti-Gα13or anti-β3 antibody. (FIG. 18F) Quantitation of integrin β3-bound talinfor FIG. 18E.

FIGS. 19A-19B demonstrate that the EEE motif of integrin β3 is importantfor mediating cell spreading on immobilized fibrinogen. (FIG. 19A)Spreading of washed human platelets pre-treated with vehicle (PBS),scramble peptide or P13 peptide on immobilized fibrinogen for 1 hour.Platelets were stained with Alexa Fluor 546-conjugated phalloidin. (FIG.19B) Microscopy images of spreading 123 cells or E to A-mutant cells onfibrinogen for 1 hour. Merged integrin β3 (green) fluorescence and talinhead (Red) fluorescence.

FIGS. 20A-20C demonstrate that the effect of P5 peptide on plateletsspreading and aggregation. (FIG. 20A) Confocal microscopy images ofDMSO, Myr-Scramble or Myr-P5 peptide treated platelets spreading onfibrinogen for 60 minutes, without or with Y27632. Merged integrin β3(green) fluorescence and Alex Fluor 546-conjugated phalloidin (Red)fluorescence. (FIG. 20B, FIG. 20C) 30 μM Myr-P5 peptide inhibited bothhuman (FIG. 20B) and mouse (FIG. 20C) platelets aggregation induced bythrombin.

FIG. 21A represents a set of Western blots using β3, talin or Gα13specific antibodies. Platelets treated with DMSO, 500 μM mP13, mP5, orcorresponding control peptides were stimulated with 0.025 U/ml thrombinat 22° C., solubilized and immunoprecipitated with anti-β₃ or preimmunerabbit serum. Lysates and immunoprecipitates were immunoblotted withanti-Gα₁₃, anti-talin or anti-β₃ antibody

FIG. 21B represents a set of graphs of a flow cytometrical analysis ofPAR4AP (50 μM)-induced Oregon-Green labelled fibrinogen binding to humanplatelets pre-treated with DMSO, 250 μM Myr-Scrambled peptide or 250 μMmP5 (upper panel), or pre-treated with DMSO, 150 μM Myr-Scrambledpeptide or 150 μM mP13 (lower panel).

FIG. 21C represent as a set of fluorescence microscopy images ofphalloidin-stained human platelets spreading on fibrinogen for 1 hr.Platelets were pre-treated with 0.05% DMSO, 250 μM myristoylated mP5,mP13, or the corresponding control peptides for 5 min at roomtemperature.

FIG. 21D represents a graph of % resting platelet adhesion toimmobilized fibrinogen of platelets treated with mP₅ or mP₁₃ (250 μM) ortheir respective scrambled peptide (mean±SD, n=3, *p<0.001).

FIG. 22 is an illustration depicting the differences in inside-out andoutside-in signaling upon vascular injury treated with a currentintegrin antagonist vs. an outside-in signal inhibitor.

FIG. 23 is an illustration depicting signal transduction pathways ofintegrins.

FIG. 24 represents a set of graphs depicting the differences in plateletATP secretion (top) and % platelet aggregation (bottom) upon treatmentwith mP5 peptide or a scambled control peptide thereof.

FIG. 25 is an illustration of a micelle.

FIG. 26 represents a graph of tail bleeding time in mice treated withIntegrillin or a saline control. Median value indicated.

FIG. 27 represents a graph of ATP secretion by platelets stimulated with0.1 U thrombin and pre-incubated with FEEERA (SEQ ID NO: 87) peptidedissolved in DMSO or the control scrambled peptide thereof.

FIG. 28 represents a graph of the occlusion time of mice treated with anEXE motif peptide (FEEERA; SEQ ID NO: 87) or the scrambled peptidecontrol (ERAFEE; SEQ ID NO: 91).

FIG. 29 represents a graph of turbidometric measurement of plateletaggregation showing the doses of the EXE motif peptide FEEERA (SEQ IDNO: 87) required for inhibition of platelet aggregation among thepeptide in micellar form vs. the peptide dissolved in DMSO.

FIG. 30A represents a table including scores of ability to inhibitplatelet aggregation for varying doses of EEERA (SEQ ID NO: 21; mP5 orthe peptide FEEERA (SEQ ID NO: 87).

FIG. 30B represents a set of graphs depicting platelet aggregationtraces for the mP5 peptide (EEERA; SEQ ID NO: 21), the scambled controlof mP5, the peptide of FEEERA (SEQ ID NO: 87), or its scrambled control.

DETAILED DESCRIPTION

Inhibitors of Binding Interactions Between a β Integrin and a G Proteinα Subunit

Provided herein are compounds that inhibit a protein-protein bindinginteraction between a β integrin and a G protein a subunit. Thecompounds of the invention may be considered as inhibitors of a βintegrin binding to a G protein a subunit and/or inhibitors of a Gprotein a subunit binding to a β integrin. In some embodiments, thecompounds are competitive binding inhibitors. In certain aspects, thecompounds bind to the site of a β integrin to which a G protein asubunit binds. In certain aspects, the compounds bind to the site of a Gprotein a subunit to which a β integrin binds. In alternativeembodiments, the compounds are non-competitive binding inhibitors. Incertain aspects, the compounds inhibit the binding interaction between aβ integrin and a G protein a subunit, yet the compounds bind to a siteof β integrin other than the site to which G protein a subunit binds orthe compounds bind to a site of a G protein a subunit other than thesite to which β integrin binds.

The inhibition provided by the compounds of the invention may not be a100% or complete inhibition or abrogation of the binding interactionbetween the β integrin and G protein a subunit. Rather, there arevarying degrees of inhibition of which one of ordinary skill in the artrecognizes as having a potential benefit or therapeutic effect. In thisrespect, the compounds of the invention may inhibit the bindinginteraction between a β integrin and a G protein a subunit to any amountor level. In exemplary embodiments, the compound provides at least orabout a 10% inhibition (e.g., at least or about a 20% inhibition, atleast or about a 30% inhibition, at least or about a 40% inhibition, atleast or about a 50% inhibition, at least or about a 60% inhibition, atleast or about a 70% inhibition, at least or about a 80% inhibition, atleast or about a 90% inhibition, at least or about a 95% inhibition, atleast or about a 98% inhibition) of the binding between a β integrin andG protein α subunit. In some embodiments, the compound completelyabrogates the binding interaction between the β integrin and the Gprotein α subunit, such that no β integrin-G protein α subunit bindingcomplexes are detectable in a sample obtained from a subject, asmeasured by, for example, immunoprecipitation, Western blotting,immunohistochemistry, and the like.

In some embodiments of the invention, the compounds inhibit the bindinginteraction between a wild-type human β integrin and a wild-type human Gprotein α subunit. In exemplary aspects, the wild-type human G protein αsubunit is a wild-type human Gα₁₂ or a wild-type human Gα₁₃. Inexemplary aspects, the wild-type human β integrin is a wildtype humanβ_(1A) integrin, wild-type human β_(1D) integrin, wild-type human β₂integrin, wildtype human β₃ integrin, wildtype human β₅ integrin,wildtype human β₆ integrin, or wildtype human β₇ integrin. The aminoacid sequences of these wild-type human proteins are known in the artand are available in the Protein database of the National Center forBiotechnology Information (NCBI) website as NCBI Reference Sequence Nos.NP_006563.2 (Gα₁₃), NP_β31379.2 (Gα₁₂), NP_002202 (β_(1A) integrin),NP_391988 (β_(1D) integrin), NP_000202 (β₂ integrin), NP_000203 (β₃integrin), NP_002204.2 (β₅ integrin), NP_000879.2 (β₆ integrin), andNP_000770.1 (β₇ integrin). The amino acid sequences also are providedherein as SEQ ID NOs: 1-4 and 10-18.

In other exemplary embodiments, the compounds inhibit the bindinginteraction between a β integrin and a G protein α subunit, wherein oneor both of the β integrin and the G protein α subunit is/arenot-wild-type, e.g., mutant. For example, the amino acid sequence of themutant β integrin differs at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, or more) positions from the amino acidsequence of a wild-type human β integrin recognized in the art (e.g.,β_(1A) integrin, β_(1D) integrin, β₂ integrin, β₃ integrin, β₅ integrin,β₆ integrin, β₇ integrin). The amino acid sequence of the mutant βintegrin in exemplary aspects is about 98% or less (e.g., about 95% orless, about 90% or less, about 85% or less, about 80% or less, about 75%or less, about 70% or less, about 65% or less, about 60% or less, about55% or less, about 50% or less) identical to that of a wild-type βintegrin. Also, for example, the amino acid sequence of the mutant Gprotein α subunit differs at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, or more) positions from the amino acidsequence of a wild-type human G protein a subunit recognized in the art(e.g., Gα₁₂, Gα₁₃). The amino acid sequence of the mutant G protein αsubunit in exemplary aspects is about 98% or less (e.g., about 95% orless, about 90% or less, about 85% or less, about 80% or less, about 75%or less, about 70% or less, about 65% or less, about 60% or less, about55% or less, about 50% or less) identical to that of a wild-type Gprotein α subunit.

In exemplary aspects, the compound is an antibody, an antibody analog, apeptide, a peptide analog (e.g., peptoid, peptidomimetic), a nucleicacid molecule encoding any of the antibodies or peptides, or analogsthereof, or a small molecule compound (e.g., small molecule compoundrationally designed based on any of the antibodies or peptides describedherein).

Antibodies and Analogs Thereof

In some embodiments of the invention, the compound that inhibits abinding interaction between a β integrin and a G protein α subunitcomprises an antibody, or antigen binding fragment thereof. In someembodiments of the invention, the compound that inhibits a bindinginteraction between a β integrin and a G protein α subunit is anantibody, or antigen binding fragment thereof. The antibody may be anytype of immunoglobulin known in the art. In exemplary embodiments, theantibody is an antibody of isotype IgA, IgD, IgE, IgG, or IgM. Also, theantibody in some embodiments is a monoclonal antibody. In otherembodiments, the antibody is a polyclonal antibody.

In some embodiments, the antibody is a naturally-occurring antibody,e.g., an antibody isolated and/or purified from a mammal, e.g., mouse,rabbit, goat, horse, chicken, hamster, human, and the like. In thisregard, the antibody may be considered as a mammalian antibody, e.g., amouse antibody, rabbit antibody, goat antibody, horse antibody, chickenantibody, hamster antibody, human antibody, and the like. Methods ofproducing naturally-occurring antibodies are known in the art, some ofwhich are described further herein under the section entitled “Methodsof Antibody Production.”

In some embodiments, the antibody is a genetically-engineered antibody,e.g., a single chain antibody, a humanized antibody, a chimericantibody, a CDR-grafted antibody, an antibody which includes portions ofCDR sequences specific for a β integrin or a G protein a subunit, ahumaneered antibody, a bispecific antibody, a trispecific antibody, andthe like. Genetic engineering techniques also provide the ability tomake fully human antibodies in a non-human source.

In some aspects, the genetically-engineered antibody is a single chainantibody (SCA) specific for a β integrin or a G protein α subunit. Inparticular aspects, the SCA binds to the site of a β integrin to which Gprotein α subunit binds or the SCA binds to the site of a G protein asubunit to which a β integrin binds. In exemplary aspects, the SCA bindsto an epitope as further described herein under the section entitled“Epitopes.” Methods of making SCAs are known in the art. See, forexample, Davis et al., Nature Biotechnology 9: 165-169 (1991).

In some aspects, the antibody is a chimeric antibody. The term “chimericantibody” is used herein to refer to an antibody containing constantdomains from one species and the variable domains from a second, or moregenerally, containing stretches of amino acid sequence from at least twospecies. In particular aspects, the chimeric antibody binds to the siteof a P integrin to which a G protein α subunit binds or the chimericantibody binds to the site of a G protein α subunit to which a βintegrin binds. In exemplary aspects, the chimeric antibody binds to anepitope as further described herein under the section entitled“Epitopes.”

In some aspects, the antibody is a humanized antibody. The term“humanized” when used in relation to antibodies refers to antibodieshaving at least CDR regions from a non-human source which are engineeredto have a structure and immunological function more similar to truehuman antibodies than the original source antibodies. For example,humanizing can involve grafting CDR from a non-human antibody, such as amouse antibody, into a human antibody. Humanizing also can involveselect amino acid substitutions to make a non-human sequence look morelike a human sequence. In particular aspects, the humanized antibodybinds to the site of a β integrin to which a G protein α subunit bindsor the humanized antibody binds to the site of a G protein α subunit towhich a β integrin binds. In exemplary aspects, the humanized antibodybinds to an epitope as further described herein under the sectionentitled “Epitopes.”

Use of the terms “chimeric or humanized” herein is not meant to bemutually exclusive, and rather, is meant to encompass chimericantibodies, humanized antibodies, and chimeric antibodies that have beenfurther humanized. Except where context otherwise indicates, statementsabout (properties of, uses of, testing of, and so on) chimericantibodies of the invention apply to humanized antibodies of theinvention, and statements about humanized antibodies of the inventionpertain also to chimeric antibodies. Likewise, except where contextdictates, such statements also should be understood to be applicable toantibodies and antigen binding fragments of such antibodies of theinvention.

In some aspects, the antibody is a CDR-grafted antibody specific for a βintegrin or a G protein α subunit. In particular aspects, theCDR-grafted antibody binds to the site of a β integrin to which a Gprotein α subunit binds or the CDR-grafted antibody binds to the site ofa G protein α subunit to which a β integrin binds. In exemplary aspects,the CDR-grafted antibody binds to an epitope as further described hereinunder the section entitled “Epitopes.” Methods of making CDR-graftedantibodies are known in the art. See, for example, Lo, Benny, AntibodyEngineering: Methods and Protocols, Volume 248 (2004), which isincorporated by reference in its entirety.

In some aspects, the antibody is a bispecific or trispecific antibodyspecific for a P3 integrin or a G protein α subunit. In particularaspects, the bispecific or trispecific antibody binds to the site of a βintegrin to which a G protein α subunit binds or the bispecific ortrispecific antibody binds to the site of a G protein α subunit to whicha β integrin binds. In exemplary aspects, the bispecific or trispecificantibody binds to an epitope as further described herein under thesection entitled “Epitopes.” Methods of making bispecific or trispecificantibodies are known in the art. See, for example, Marvin and Zhu, ActaPharmacologica Sinica 26: 649-658 (2005) and U.S. Pat. No. 6,551,592.

In some aspects, the antibody is a Humaneered™ antibody. Humaneeringtechnology is a proprietary method of KaloBios Pharmaceuticals, Inc.(San Francisco, Calif.) for converting non human antibodies intoengineered human antibodies. Humaneered™ antibodies are high affinity,and highly similar to human germline antibody sequences.

In some embodiments, the antibody has a level of affinity or avidity forthe β integrin which is sufficient to prevent the G protein α subunitfrom binding to the β integrin. In some embodiments, the antibody has alevel of affinity or avidity for the G protein α subunit which issufficient to prevent a β integrin from binding G protein α subunit.Therefore in some embodiments, the affinity constant, K_(a), (which isthe inverted dissociation constant, K_(d)) of the antibody of theinvention for the a β integrin is greater than the K_(a) of G protein αsubunit for the β integrin. Alternatively, in some embodiments, theK_(a) of the antibody of the invention for the G protein α subunit isgreater than that of the β integrin for the G protein α subunit. Bindingconstants, including dissociation constants, may be determined bymethods known in the art, including, for example, methods which utilizethe principles of surface plasmon resonance, e.g., methods utilizing aBiacore™ system.

In some embodiments, the antibody is in monomeric form, while in otherembodiments, the antibody is conjugated to one or more antibodies (e.g.,each of which recognize the same epitope of the first antibody).Accordingly, in some aspects, the antibody is in polymeric, oligomeric,or multimeric form. In certain embodiments in which the antibodycomprises two or more distinct antigen binding regions fragments, theantibody is considered bispecific, trispecific, or multi-specific, orbivalent, trivalent, or multivalent, depending on the number of distinctepitopes that are recognized and bound by the antibody.

Antigen Binding Fragments

In some aspects of the invention, the compound which inhibits a bindinginteraction between an a β integrin and a G protein α subunit is anantigen binding fragment of an antibody. The antigen binding fragment(also referred to herein as “antigen binding portion”) may be an antigenbinding fragment of any of the antibodies described herein. The antigenbinding fragment can be any part of an antibody that has at least oneantigen binding site, including, but not limited to, Fab, F(ab′)₂, dsFv,sFv, diabodies, triabodies, bis-scFvs, fragments expressed by a Fabexpression library, domain antibodies, VhH domains, V-NAR domains, VHdomains, VL domains, and the like. Antibody fragments of the invention,however, are not limited to these exemplary types of antibody fragments.

A domain antibody comprises a functional binding unit of an antibody,and can correspond to the variable regions of either the heavy (V_(H))or light (V_(L)) chains of antibodies. A domain antibody can have amolecular weight of approximately 13 kDa, or approximately one-tenth ofa full antibody. Domain antibodies may be derived from full antibodiessuch as those described herein. The antigen binding fragments in someembodiments are monomeric or polymeric, bispecific or trispecific,bivalent or trivalent.

Antibody fragments that contain the antigen binding, or idiotype, of theantibody molecule may be generated by techniques known in the art. Forexample, such fragments include, but are not limited to, the F(ab′)₂fragment which may be produced by pepsin digestion of the antibodymolecule; the Fab′ fragments which may be generated by reducing thedisulfide bridges of the F(ab′)₂ fragment, and the two Fab′ fragmentswhich may be generated by treating the antibody molecule with papain anda reducing agent.

A single-chain variable region fragment (sFv) antibody fragment, whichconsists of a truncated Fab fragment comprising the variable (V) domainof an antibody heavy chain linked to a V domain of a light antibodychain via a synthetic peptide, can be generated using routinerecombinant DNA technology techniques (see, e.g., Janeway et al.,supra). Similarly, disulfide-stabilized variable region fragments (dsFv)can be prepared by recombinant DNA technology (see, e.g., Reiter et al.,Protein Engineering, 7, 697-704 (1994)).

Recombinant antibody fragments, e.g., scFvs, can also be engineered toassemble into stable multimeric oligomers of high binding avidity andspecificity to different target antigens. Such diabodies (dimers),triabodies (trimers) or tetrabodies (tetramers) are well known in theart, see e.g., Kortt et al., Biomol Eng. 2001 18:95-108, (2001) andTodorovska et al., J Immunol Methods. 248:47-66, (2001).

Bispecific antibodies (bscAb) are molecules comprising two single-chainFv fragments joined via a glycine-serine linker using recombinantmethods. The V light-chain (V_(L)) and V heavy-chain (V_(H)) domains oftwo antibodies of interest in exemplary embodiments are isolated usingstandard PCR methods. The V_(L) and V_(H) cDNA's obtained from eachhybridoma are then joined to form a single-chain fragment in a two-stepfusion PCR. Bispecific fusion proteins are prepared in a similar manner.Bispecific single-chain antibodies and bispecific fusion proteins areantibody substances included within the scope of the present invention.Exemplary bispecific antibodies are taught in U.S. Patent ApplicationPublication No. 2005-0282233A1 and International Patent ApplicationPublication No. WO 2005/087812, both applications of which areincorporated herein by reference in their entirety.

Methods of Antibody or Antigen Binding Fragment Production

Suitable methods of making antibodies are known in the art. Forinstance, standard hybridoma methods are described in, e.g., Harlow andLane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988), and CA.Janeway et al. (eds.), Immunobiology, 5^(th) Ed., Garland Publishing,New York, N.Y. (2001)).

Briefly, a polyclonal antibody is prepared by immunizing an animal withan immunogen comprising a polypeptide of the present invention andcollecting antisera from that immunized animal. A wide range of animalspecies can be used for the production of antisera. In some aspects, ananimal used for production of anti-antisera is a non-human animalincluding rabbits, mice, rats, hamsters, goat, sheep, pigs or horses.Because of the relatively large blood volume of rabbits, a rabbit is apreferred choice for production of polyclonal antibodies. In anexemplary method for generating a polyclonal antisera immunoreactivewith the chosen β integrin epitope, 50 μg of β integrin antigen isemulsified in Freund's Complete Adjuvant for immunization of rabbits. Atintervals of, for example, 21 days, 50 μg of epitope are emulsified inFreund's Incomplete Adjuvant for boosts. Polyclonal antisera may beobtained, after allowing time for antibody generation, simply bybleeding the animal and preparing serum samples from the whole blood.

Monoclonal antibodies for use in the invention may be prepared using anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture. These include but are not limited tothe hybridoma technique originally described by Koehler and Milstein(Nature 256: 495-497, 1975), the human B-cell hybridoma technique(Kosbor et al., Immunol Today 4:72, 1983; Cote et al., Proc Natl AcadSci 80: 2026-2030, 1983) and the EBV-hybridoma technique (Cole et al.,Monoclonal Antibodies and Cancer Therapy, Alan R Liss Inc, New YorkN.Y., pp 77-96, (1985).

Briefly, in exemplary embodiments, to generate monoclonal antibodies, amouse is injected periodically with recombinant β integrin against whichthe antibody is to be raised (e.g., 10-20 μg emulsified in Freund'sComplete Adjuvant). The mouse is given a final pre-fusion boost of an βintegrin polypeptide in PBS, and four days later the mouse is sacrificedand its spleen removed. The spleen is placed in 10 ml serum-free RPMI1640, and a single cell suspension is formed by grinding the spleenbetween the frosted ends of two glass microscope slides submerged inserum-free RPMI 1640, supplemented with 2 mM L-glutamine, 1 mM sodiumpyruvate, 100 units/ml penicillin, and 100 μg/ml streptomycin (RPMI)(Gibco, Canada). The cell suspension is filtered through sterile 70-meshNitex cell strainer (Becton Dickinson, Parsippany, N.J.), and is washedtwice by centrifuging at 200 g for 5 minutes and resuspending the pelletin 20 ml serum-free RPMI. Splenocytes taken from three naive Balb/c miceare prepared in a similar manner and used as a control. NS-1 myelomacells, kept in log phase in RPMI with 11% fetal bovine serum (FBS)(Hyclone Laboratories, Inc., Logan, Utah) for three days prior tofusion, are centrifuged at 200 g for 5 minutes, and the pellet is washedtwice.

Spleen cells (1×10⁸) are combined with 2.0×10⁷ NS-1 cells andcentrifuged, and the supernatant is aspirated. The cell pellet isdislodged by tapping the tube, and 1 ml of 37° C. PEG 1500 (50% in 75 mMHepes, pH 8.0) (Boehringer Mannheim) is added with stirring over thecourse of 1 minute, followed by the addition of 7 ml of serum-free RPMIover 7 minutes. An additional 8 ml RPMI is added and the cells arecentrifuged at 200 g for 10 minutes. After discarding the supernatant,the pellet is resuspended in 200 ml RPMI containing 15% FBS, 100 μMsodium hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine (HAT) (Gibco),25 units/ml IL-6 (Boehringer Mannheim) and 1.5×10⁶ splenocytes/ml andplated into 10 Corning flat-bottom 96-well tissue culture plates(Corning, Corning N.Y.).

On days 2, 4, and 6, after the fusion, 100 μl of medium is removed fromthe wells of the fusion plates and replaced with fresh medium. On day 8,the fusion is screened by ELISA, testing for the presence of mouse IgGbinding to β integrin as follows. Immulon 4 plates (Dynatech, Cambridge,Mass.) are coated for 2 hours at 37° C. with 100 ng/well of β integrindiluted in 25 mM Tris, pH 7.5. The coating solution is aspirated and 200μl/well of blocking solution (0.5% fish skin gelatin (Sigma) diluted inCMF-PBS) is added and incubated for 30 min. at 37° C. Plates are washedthree times with PBS with 0.05% Tween 20 (PBST) and 50 l culturesupernatant is added. After incubation at 37° C. for 30 minutes, andwashing as above, 50 μl of horseradish peroxidase conjugated goatanti-mouse IgG(fc) (Jackson ImmunoResearch, West Grove, Pa.) diluted1:3500 in PBST is added. Plates are incubated as above, washed fourtimes with PBST, and 100 μl substrate, consisting of 1 mg/ml o-phenylenediamine (Sigma) and 0.1 μl/ml 30% H₂O₂ in 100 mM Citrate, pH 4.5, areadded. The color reaction is stopped after 5 minutes with the additionof 50 μl of 15% H₂SO₄. A₄₉₀ is read on a plate reader (Dynatech).

Selected fusion wells are cloned twice by dilution into 96-well platesand visual scoring of the number of colonies/well after 5 days. Themonoclonal antibodies produced by hybridomas are isotyped using theIsostrip system (Boehringer Mannheim, Indianapolis, Ind.).

When the hybridoma technique is employed, myeloma cell lines may beused. Such cell lines suited for use in hybridoma-producing fusionprocedures preferably are non-antibody-producing, have high fusionefficiency, and enzyme deficiencies that render them incapable ofgrowing in certain selective media which support the growth of only thedesired fused cells (hybridomas). For example, where the immunizedanimal is a mouse, one may use P3-X63/Ag8, P3-X63-Ag8.653, NS1/1.Ag 4 1,Sp210-Agl4, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/15XX0 Bul; forrats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266,GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection withcell fusions. It should be noted that the hybridomas and cell linesproduced by such techniques for producing the monoclonal antibodies arecontemplated to be novel compositions of the invention.

Depending on the host species, various adjuvants may be used to increaseimmunological response. Such adjuvants include but are not limited toFreund's, mineral gels such as aluminum hydroxide, and surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG(bacilli Calmette-Guerin) and Corynebacterium parvum are potentiallyuseful human adjuvants.

Alternatively, other methods, such as EBV-hybridoma methods (Haskard andArcher, J. Immunol. Methods, 74(2), 361-67 (1984),and Roder et al.₅Methods Enzymol., 121, 140-67 (1986)), and bacteriophage vectorexpression systems (see, e.g., Huse et al., Science, 246, 1275-81(1989)) are known in the art. Further, methods of producing antibodiesin non-human animals are described in, e.g., U.S. Pat. Nos. 5,545,806,5,569,825, and 5,714,352, and U.S. Patent Application Publication No.2002/0197266 A1).

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inOrlandi et al (Proc Natl Acad Sci 86: 3833-3837; 1989), and Winter G andMilstein C (Nature 349: 293-299, 1991).

Phage display furthermore can be used to generate the antibody of theinvention. In this regard, phage libraries encoding antigen-bindingvariable (V) domains of antibodies can be generated using standardmolecular biology and recombinant DNA techniques (see, e.g., Sambrook etal. (eds.), Molecular Cloning, A Laboratory Manual, 3^(rd) Edition, ColdSpring Harbor Laboratory Press, New York (2001)), Phage encoding avariable region with the desired specificity are selected for specificbinding to the desired antigen, and a complete or partial antibody isreconstituted comprising the selected variable domain. Nucleic acidsequences encoding the reconstituted antibody are introduced into asuitable cell line, such as a myeloma cell used for hybridomaproduction, such that antibodies having the characteristics ofmonoclonal antibodies are secreted by the cell (see, e.g., Janeway etal., supra, Huse et al., supra, and U.S. Pat. No. 6,265,150). Relatedmethods also are described in U.S. Pat. Nos. 5,403,484; 5,571,698;5,837,500; 5,702,892. The techniques described in U.S. Pat. Nos.5,780,279; 5,821,047; 5,824,520; 5,855,885; 5,858,657; 5,871,907;5,969,108; 6,057,098; 6,225,447,

Antibodies can be produced by transgenic mice that are transgenic forspecific heavy and light chain immunoglobulin genes. Such methods areknown in the art and described in, for example U.S. Pat. Nos. 5,545,806and 5,569,825, and Janeway et al., supra.

Methods for generating humanized antibodies are well known in the artand are described in detail in, for example, Janeway et al., supra, U.S.Pat. Nos. 5,225,539, 5,585,089 and 5,693,761, European Patent No.0239400 B1, and United Kingdom Patent No. 2188638. Humanized antibodiescan also be generated using the antibody resurfacing technologydescribed in U.S. Pat. No. 5,639,641 and Pedersen et al., J. Mol. Biol,235, 959-973 (1994).

Techniques developed for the production of “chimeric antibodies”, thesplicing of mouse antibody genes to human antibody genes to obtain amolecule with appropriate antigen specificity and biological activity,can be used (Morrison et al., Proc Natl Acad Sci 81: 6851-6855, 1984;Neuberger et al., Nature 312: 604-608, 1984; Takeda et al., Nature 314:452-454; 1985). Alternatively, techniques described for the productionof single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted toproduce β integrin—or G protein α subunit-specific single chainantibodies.

A preferred chimeric or humanized antibody has a human constant region,while the variable region, or at least a CDR, of the antibody is derivedfrom a non-human species. Methods for humanizing non-human antibodiesare well known in the art. (see U.S. Pat. Nos. 5,585,089, and5,693,762). Generally, a humanized antibody has one or more amino acidresidues introduced into its framework region from a source which isnon-human. Humanization can be performed, for example, using methodsdescribed in Jones et al. (Nature 321: 522-525, 1986), Riechmann et al.,(Nature, 332: 323-327, 1988) and Verhoeyen et al. (Science239:1534-1536, 1988), by substituting at least a portion of a rodentcomplementarity-determining region (CDRs) for the corresponding regionsof a human antibody. Numerous techniques for preparing engineeredantibodies are described, e.g., in Owens and Young, J. Immunol. Meth.,168:149-165 (1994). Further changes can then be introduced into theantibody framework to modulate affinity or immunogenicity.

Likewise, using techniques known in the art to isolate CDRs,compositions comprising CDRs are generated. Complementarity determiningregions are characterized by six polypeptide loops, three loops for eachof the heavy or light chain variable regions. The amino acid position ina CDR is defined by Kabat et al., “Sequences of Proteins ofImmunological Interest,” U.S. Department of Health and Human Services,(1983), which is incorporated herein by reference. For example,hypervariable regions of human antibodies are roughly defined to befound at residues 28 to 35, from 49-59 and from residues 92-103 of theheavy and light chain variable regions (Janeway and Travers,Immunobiology, 2^(nd) Edition, Garland Publishing, New York, (1996)).The murine CDR also are found at approximately these amino acidresidues. It is understood in the art that CDR regions may be foundwithin several amino acids of these approximated residues set forthabove. An immunoglobulin variable region also consists of four“framework” regions surrounding the CDRs (FR1-4). The sequences of theframework regions of different light or heavy chains are highlyconserved within a species, and are also conserved between human andmurine sequences.

Compositions comprising one, two, and/or three CDRs of a heavy chainvariable region or a light chain variable region of a monoclonalantibody are generated. Techniques for cloning and expressing nucleotideand polypeptide sequences are well-established in the art (see e.g.Sambrook et al., Molecular Cloning: A Laboratory Manual, 2^(nd) Edition,Cold Spring Harbor, N.Y. (1989)). The amplified CDR sequences areligated into an appropriate plasmid. The plasmid comprising one, two,three, four, five and/or six cloned CDRs optionally contains additionalpolypeptide encoding regions linked to the CDR.

Framework regions (FR) of a murine antibody are humanized bysubstituting compatible human framework regions chosen from a largedatabase of human antibody variable sequences, including over twelvehundred human V_(H) sequences and over one thousand V_(L) sequences. Thedatabase of antibody sequences used for comparison is downloaded fromAndrew C. R. Martin's KabatMan web page(http://www.rubic.rdg.ac.uk/abs/). The Kabat method for identifying CDRprovides a means for delineating the approximate CDR and frameworkregions from any human antibody and comparing the sequence of a murineantibody for similarity to determine the CDRs and FRs. Best matchedhuman V_(H) and V_(L) sequences are chosen on the basis of high overallframework matching, similar CDR length, and minimal mismatching ofcanonical and V_(H)/V_(L) contact residues. Human framework regions mostsimilar to the murine sequence are inserted between the murine CDR.Alternatively, the murine framework region may be modified by makingamino acid substitutions of all or part of the native framework regionthat more closely resemble a framework region of a human antibody.

Additionally, another useful technique for generating antibodies for usein the present invention may be one which uses a rational design typeapproach. The goal of rational design is to produce structural analogsof biologically active polypeptides or compounds with which theyinteract (agonists, antagonists, inhibitors, peptidomimetics, bindingpartners, etc.). In one approach, one would generate a three-dimensionalstructure for the antibodies or an epitope binding fragment thereof.This could be accomplished by x-ray crystallography, computer modelingor by a combination of both approaches. An alternative approach,“alanine scan,” involves the random replacement of residues throughoutmolecule with alanine, and the resulting affect on function determined.

It also is possible to solve the crystal structure of the specificantibodies. In principle, this approach yields a pharmacore upon whichsubsequent drug design can be based. It is possible to bypass proteincrystallography altogether by generating anti-idiotypic antibodies to afunctional, pharmacologically active antibody. As a mirror image of amirror image, the binding site of anti-idiotype would be expected to bean analog of the original antigen. The anti-idiotype could then be usedto identify and isolate additional antibodies from banks of chemically-or biologically-produced peptides.

Chemically constructed bispecific antibodies may be prepared bychemically cross-linking heterologous Fab or F(ab′)₂ fragments by meansof chemicals such as heterobifunctional reagentsuccinimidyl-3-(2-pyridyldithiol)-propionate (SPDP, Pierce Chemicals,Rockford, Ill.). The Fab and F(ab′)₂ fragments can be obtained fromintact antibody by digesting it with papain or pepsin, respectively(Karpovsky et al., J. Exp. Med. 160:1686-701, 1984; Titus et al., J.Immunol., 138:4018-22, 1987).

Methods of testing antibodies for the ability to bind to the epitope ofthe β integrin regardless of how the antibodies are produced are knownin the art and include any antibody-antigen binding assay, such as, forexample, radioimmunoassay (RIA), ELISA, Western blot,immunoprecipitation, and competitive inhibition assays (see, e.g.,Janeway et al., infra, and U.S. Patent Application Publication No.2002/0197266 A1).

Aptamers

In some embodiments, the compound that inhibits a binding interactionbetween β integrin and G protein α subunit is an analog of an antibody.In some aspects, the compound is an aptamer. Recent advances in thefield of combinatorial sciences have identified short polymer sequences(e.g., oligonucleic acid or peptide molecules) with high affinity andspecificity to a given target. For example, SELEX technology has beenused to identify DNA and RNA aptamers with binding properties that rivalmammalian antibodies, the field of immunology has generated and isolatedantibodies or antibody fragments which bind to a myriad of compounds andphage display has been utilized to discover new peptide sequences withvery favorable binding properties. Based on the success of thesemolecular evolution techniques, it is certain that molecules can becreated which bind to any target molecule. A loop structure is ofteninvolved with providing the desired binding attributes as in the caseof: aptamers which often utilize hairpin loops created from shortregions without complimentary base pairing, naturally derived antibodiesthat utilize combinatorial arrangement of looped hyper-variable regionsand new phage display libraries utilizing cyclic peptides that haveshown improved results when compared to linear peptide phage displayresults. Thus, sufficient evidence has been generated to suggest thathigh affinity ligands can be created and identified by combinatorialmolecular evolution techniques. For the invention, molecular evolutiontechniques can be used to isolate compounds specific for the β integrinsor G protein α subunits described herein that inhibit the bindinginteraction between β integrin and G protein α subunit. For more onaptamers, see, generally, Gold, L., Singer, B., He, Y. Y., Brody. E.,“Aptamers As Therapeutic And Diagnostic Agents,” J. Biotechnol. 74:5-13(2000). Relevant techniques for generating aptamers may be found in U.S.Pat. No. 6,699,843, which is incorporated by reference in its entirety.

Epitopes

By “epitope” as used herein is meant the region of or within the βintegrin or G protein α subunit which is bound by the compound, e.g.,the antibody, the antigen binding fragment, the aptamer. In someembodiments, the epitope is a linear epitope. By “linear epitope” asused herein refers to the region of or within the β integrin or Gprotein α subunit which is bound by the compound, which region iscomposed of contiguous amino acids of the amino acid sequence of the βintegrin or G protein α subunit. The amino acids of a linear epitope arelocated in close proximity to each other in the primary structure of theantigen and the secondary and/or tertiary structure(s) of the antigen.For example, when the antigen, e.g., β integrin or G protein α subunit,is in its properly folded state (e.g., its native conformation), thecontiguous amino acids of the linear epitope are located in closeproximity to one another.

In other aspects, the epitope of the binding construct is aconformational epitope. By “conformational epitope” is meant an epitopewhich is composed of amino acids which are located in close proximity toone another only when the β integrin or G protein α subunit is in itsproperly folded state, but are not contiguous amino acids of the aminoacid sequence of the β integrin or G protein α subunit.

In exemplary embodiments of the invention, the compound that inhibits abinding interaction between a β integrin and a G protein α subunit bindsto an epitope of a β integrin. In some aspects, the epitope to which thecompound binds is within the cytoplasmic domain of a P integrin. Inexemplary aspects, the epitope to which the compound binds is within thecytoplasmic domain of a β_(1A) integrin, β_(1D) integrin, β₂ integrin,β₃ integrin, β₅ integrin, β₆ integrin, or β₇ integrin. In exemplaryaspects, the epitope to which the compound binds is within amino acids738-777 of a β_(1A) integrin (SEQ ID NO: 12) or amino acids 732-778 of aβ_(1A) integrin (SEQ ID NO: 12). In exemplary aspects, the epitope towhich the compound binds is within amino acids 738-776 of a β_(1D)integrin (SEQ ID NO: 13) or amino acids 732-781 of a β_(1D) integrin(SEQ ID NO: 13). In exemplary aspects, the epitope to which the compoundbinds is within amino acids 702-746 of a β₂ integrin (SEQ ID NO: 14) oramino acids 702-747 of a β₂ integrin (SEQ ID NO: 14). In exemplaryaspects, the epitope to which the compound binds is within amino acids722-761 of a β₃ integrin (SEQ ID NO: 15) or amino acids 716-762 of a β₃integrin (SEQ ID NO: 15). In exemplary aspects, the epitope to which thecompound binds is within amino acids 720-765 of a β₅ integrin (SEQ IDNO: 16) or amino acids 720-776 of a β₅ integrin (SEQ ID NO: 16). Inexemplary aspects, the epitope to which the compound binds is withinamino acids 710-755 of a β₆ integrin (SEQ ID NO: 17) or amino acids710-767 of a β₆ integrin (SEQ ID NO: 17). In exemplary aspects, theepitope to which the compound binds is within amino acids 728-773 of aβ₇ integrin (SEQ ID NO: 18) or amino acids 728-779 of a β₇ integrin (SEQID NO: 18).

In exemplary embodiments of the invention, the compound that inhibits abinding interaction between a β integrin and a G protein α subunit bindsto an epitope of the G protein a subunit. In some aspects, the epitopeto which the compound binds is within the Switch Region I of a G proteinα subunit. In exemplary aspects, the epitope to which the compound bindsis within the Switch Region I of G protein α subunit Gα₁₁₂ or Gα₁₃. Inexemplary aspects, the compound binds to an epitope within amino acids201-216 of Gα₁₂ (SEQ ID NO: 11) or amino acids 197-212 of Gα₁₃ (SEQ IDNO: 10). In exemplary aspects, the compound binds to an epitope withinamino acids 197-209 of Gα₁₃ (SEQ ID NO: 10) or amino acids 198-206 ofGα₁₃ (SEQ ID NO: 10). In exemplary aspects, the compound binds to anepitope within amino acids 203-211 of Gα₁₂ (SEQ ID NO: 11).

In yet other embodiments, the compound that inhibits the bindinginteraction between a β integrin and a G protein α subunit binds to anepitope comprising the amino acid sequence of any of the peptides orpeptide analogs described herein. See, e.g., the section entitled“Fragments of β Integrin or G protein α Subunit and DerivativesThereof.” In exemplary aspects, the compound that inhibits the bindinginteraction between a β integrin and a G protein a subunit binds to anepitope comprising an amino acid sequence of any of SEQ ID NOs: 19-40.

Peptides

In some embodiments of the invention, the compound that inhibits abinding interaction between a β integrin and a G protein α subunit is apeptide comprising at least four amino acids connected via peptidebonds. Accordingly, the invention provides a peptide. In some aspects,the peptide is about 4 to about 50 amino acids in length. In someaspects, the compound is about 5 to about 25 amino acids in length. Insome aspects, the compound is about 5 to 20 amino acids in length. Insome aspects, the peptide is 5-15 amino acids in length. In someaspects, the peptide is 5-9 or 5-8 or 5-7 amino acids in length. In someembodiments, the peptide is a 5-mer, 6-mer, 7-mer, 8-mer, 9-mer-10-mer,11-mer, 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17-mer, 18-mer, 19-mer,or 20-mer.

Fragments Off Integrin or G Protein α Subunit and Derivatives Thereof

In some embodiments, the peptide that inhibits a binding interactionbetween a β integrin and a G protein α subunit comprises a fragment oris a fragment of a human wild-type β integrin, e.g., any of thosedisclosed herein. As used herein, the term “fragment” does not encompassa full length β integrin or a full length G protein α subunit. In someaspects, the compound comprises or is a fragment of a β_(1A) integrin,β_(1D) integrin, β₂ integrin, β₃ integrin, β₅ integrin, β₆ integrin, orβ₇ integrin. In specific aspects, the compound comprises 4 to 50 (e.g.,5 to 25) consecutive amino acids of a cytoplasmic domain of a βintegrin.

In exemplary embodiments of the invention, the compound that inhibits abinding interaction between a β integrin and a G protein α subunitcomprises 4 to 50 (e.g., 5 to 25) consecutive amino acids of thecytoplasmic domain of a β_(1A) integrin, β_(1D) integrin, β₂ integrin,β₃ integrin, β₅ integrin, β₆ integrin, or β₇ integrin. In exemplaryaspects, the compound comprises 4 to 50 (e.g., 5 to 25) consecutiveamino acids of amino acids 738-777 of a β_(1A) integrin (SEQ ID NO: 12)or amino acids 732-778 of a β_(1A) integrin (SEQ ID NO: 12). Inexemplary aspects, the compound comprises 4 to 50 (e.g., 5 to 25)consecutive amino acids of amino acids 738-776 of a β_(1D) integrin (SEQID NO: 13) or amino acids 732-781 of a β_(1D) integrin (SEQ ID NO: 13).In exemplary aspects, the compound comprises 4 to 50 (e.g., 5 to 25)consecutive amino acids of amino acids 702-746 of a β₂ integrin (SEQ IDNO: 14) or amino acids 702-747 of a β₂ integrin (SEQ ID NO: 14). Inexemplary aspects, the compound comprises 4 to 50 (e.g., 5 to 25)consecutive amino acids of amino acids 722-761 of a β₃ integrin (SEQ IDNO: 15) or amino acids 716-762 of a β₃ integrin (SEQ ID NO: 15). Inexemplary aspects, the compound comprises 4 to 50 (e.g., 5 to 25)consecutive amino acids of amino acids 720-765 of a β₅ integrin (SEQ IDNO: 16) or amino acids 720-776 of a β₅ integrin (SEQ ID NO: 16). Inexemplary aspects, the compound comprises 4 to 50 (e.g., 5 to 25)consecutive amino acids of amino acids 710-755 of a β₆ integrin (SEQ IDNO: 17) or amino acids 710-767 of a β₆ integrin (SEQ ID NO: 17). Inexemplary aspects, the compound comprises 4 to 50 (e.g., 5 to 25)consecutive amino acids of amino acids 728-773 of a β₇ integrin (SEQ IDNO: 18) or amino acids 728-779 of a β₇ integrin (SEQ ID NO: 18).

In exemplary embodiments of the invention, the compound that inhibits abinding interaction between a β integrin and a G protein α subunit thecompound comprises 4 to 50 (e.g., 5 to 25) consecutive amino acids ofthe G protein α subunit. In some aspects, the compound comprises 4 to 50(e.g., 5 to 25) consecutive amino acids of the Switch Region I of a Gprotein a subunit. In exemplary aspects, the compound comprises 4 to 50(e.g., 5 to 25) consecutive amino acids of the Switch Region I of Gprotein α subunit Gα₁₂ or Gα₁₃. In exemplary aspects, the compoundcomprises 4 to 50 (e.g., 5 to 25) consecutive amino acids of amino acids201-216 of Gα₁₂ (SEQ ID NO: 11) or amino acids 197-212 of Gα₁₃ (SEQ IDNO: 10). In exemplary aspects, the compound comprises 4 to 50 (e.g., 5to 25) consecutive amino acids of amino acids 197-209 of Gα₁₃ (SEQ IDNO: 10) or amino acids 198-206 of Gα₁₃ (SEQ ID NO: 10). In exemplaryaspects, the compound comprises 4 to 50 (e.g., 5 to 25) consecutiveamino acids of amino acids 203-211 of Gα₁₂ (SEQ ID NO: 11).

In exemplary aspects, the compound comprises a core sequence of threeamino acids which is a portion or fragment of a cytoplasmic domain ofthe β integrin. For example, the compound comprises a core sequenceidentical to amino acids 731 to 733 of the amino acid sequence of β₃integrin (SEQ ID NO: 15), which is EEE. In exemplary aspects, thecompound comprises a core sequence identical to amino acids 747-749 ofthe amino acid sequence of β_(1A) integrin (SEQ ID NO: 12) or β_(1D)integrin (SEQ ID NO: 13), amino acids 717-719 of the amino acid sequenceof β₂ integrin (SEQ ID NO: 14), or amino acids 743-745 of the amino acidsequence of β₇ integrin (SEQ ID NO: 18), each of which is EKE. Inexemplary aspects, the compound comprises a core sequence identicalamino acids 725-727 of the amino acid sequence of β₆ integrin (SEQ IDNO: 17), which is EAE. In exemplary aspects, the compound comprises acore sequence identical amino acids 735-737 of the amino acid sequenceof β₅ integrin (SEQ ID NO: 16), which is QSE.

In exemplary embodiments, the compound comprises additional amino acidsN-terminal and/or C-terminal to the core sequence. The additional aminoacids, e.g., the non-core sequence(s), may represent amino acids whichare N-terminal and/or C-terminal to the core sequence of the amino acidsequence of the β integrin. For example, the compound may comprise acore sequence of EEE (amino acids 731 to 733 of the amino acid sequenceof 33 integrin (SEQ ID NO: 15) and may additionally comprise anN-terminal non-core sequence comprising KF (Lys-Phe) and/or a C-terminalnon-core sequence comprising RA (Arg-Ala). Accordingly, the compound inexemplary aspects comprises the amino acid sequence of KFEEE (SEQ ID NO:19), KFEEERA (SEQ ID NO: 20), EEERA (SEQ ID NO: 21). In exemplaryaspects, the compound comprises an amino acid sequence of KFEEERARAKWDT(SEQ ID NO: 22).

In exemplary aspects, the compound comprises a core sequence of EKE andcomprises a N-terminal non-core sequence comprising KF (Lys-Phe) or RF(Arg-Phe) and/or a C-terminal non-core sequence comprising KM (Lys-Met),KL (Lys-Leu), or QQ (Gin-Gin). Accordingly, the compound in exemplaryaspects comprises the amino acid sequence of KFEKE (SEQ ID NO: 23),RFEKE (SEQ ID NO: 24), KFEKEKM (SEQ ID NO: 25), KFEKEKL (SEQ ID NO: 26),KFEKEQQ (SEQ ID NO: 27), RFEKEKM (SEQ ID NO: 28), RKFEKEKL (SEQ ID NO:29), RFEKEQQ (SEQ ID NO: 30), EKEKM (SEQ ID NO: 31), EKEKL (SEQ ID NO:32), or EKEQQ (SEQ ID NO: 33).

In exemplary aspects, the compound comprises a core sequence of EAE andcomprises a N-terminal non-core sequence comprising KF (Lys-Phe) and/ora C-terminal non-core sequence comprising RS (Arg-Ser). Accordingly, thecompound in exemplary aspects comprises the amino acid sequence of KFEAE(SEQ ID NO: 34), KFEAERS (SEQ ID NO: 35), or EAERS (SEQ ID NO: 36).

In exemplary aspects, the compound comprises a core sequence of QSE andcomprises a N-terminal non-core sequence comprising KF (Lys-Phe) and/ora C-terminal non-core sequence comprising RS (Arg-Ser). Accordingly, thecompound in exemplary aspects comprises the amino acid sequence of KFQSE(SEQ ID NO: 37), KFQSERS (SEQ ID NO: 38), or QSERS (SEQ ID NO: 39).

In alternative or additional embodiments, the compound comprises anon-core sequence which is not based on the wild-type sequence of the βintegrin. In exemplary aspects, the the compound may comprise a coresequence of EEE (amino acids 731 to 733 of the amino acid sequence of β₃integrin (SEQ ID NO: 15) and may additionally comprise a N-terminalnon-core sequence other than KF (Lys-Phe) and/or a C-terminal non-coresequence other than RA (Arg-Ala).

In exemplary embodiments, the peptide that inhibits a bindinginteraction between a β integrin and a G protein α subunit comprises afragment or is a fragment of a human wild-type G protein α subunit,e.g., Gα₁₂, Gα₁₃. In some aspects, the compound comprises or is afragment of Gα₁₂ or Gα₁₃. In specific aspects, the compound comprises 4to 50 (e.g., 5 to 25) consecutive amino acids of the Switch Region I ofthe G protein α subunit.

In exemplary aspects, the compound comprises 4 to 50 (e.g., 5 to 25)consecutive amino acids of the Switch Region I of G protein α subunitGα₁₁₂ or Gα₁₃. In exemplary aspects, the compound comprises 4 to 50(e.g., 5 to 25) consecutive amino acids of amino acids 201-216 of Gα₁₂(SEQ ID NO: 11) or amino acids 197-212 of Gα₁₃ (SEQ ID NO: 10). Inexemplary aspects, the compound comprises 4 to 50 (e.g., 5 to 25)consecutive amino acids of amino acids 197-209 of Gα₁₃ (SEQ ID NO: 10)or amino acids 198-206 of Gα₁₃ (SEQ ID NO: 10). In exemplary aspects,the compound comprises 4 to 50 (e.g., 5 to 25) consecutive amino acidsof amino acids 203-211 of Gα₁₂ (SEQ ID NO: 11). In some aspects, thecompound comprises an amino acid sequence of LLARRPTKGIHEY (SEQ ID NO:40).

In some embodiments, the peptide that inhibits a binding interactionbetween a β integrin and a G protein α subunit comprises an amino acidsequence which is based on the amino acid sequence of a human wild-typeβ integrin, or a fragment thereof, but differs at one or more (e.g.,two, three, four, five, six, seven, eight, nine, ten, or more) aminoacid positions, when aligned with the human wild-type β integrinsequence, or fragment thereof.

In some embodiments, the peptide that inhibits a binding interactionbetween an a β integrin and a G protein α subunit comprises an aminoacid sequence which has at least 25% sequence identity to the amino acidsequence of a human wild-type β integrin, e.g., SEQ ID NO: 15, or afragment thereof (e.g., a fragment of about 4 to about 50 contiguousamino acids of SEQ ID NO: 15). In some embodiments, the compoundcomprises an amino acid sequence which is at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or has greater than 95% sequenceidentity to SEQ ID NO: 15, or a fragment thereof (e.g., a fragment ofabout 4 to about 25 contiguous amino acids of SEQ ID NO: 15). In someembodiments, the compound comprises an amino acid sequence which is atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, orhas greater than 95% sequence identity to any of SEQ ID NOs: 12-18, or afragment thereof (e.g., a fragment of about 4 to about 25 contiguousamino acids of any one of SEQ ID NOs: 12-18). In some embodiments, thecompound comprises an amino acid sequence which is at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or has greater than95% sequence identity to any one of SEQ ID NOs: 19-39.

In some embodiments, the peptide that inhibits a binding interactionbetween a β integrin and a G protein α subunit comprises an amino acidsequence which is based on the amino acid sequence of a human wild-typeG protein α subunit, or a fragment thereof, but differs at one or more(e.g., two, three, four, five, six, seven, eight, nine, ten, or more)amino acid positions, when aligned with the human wild-type G protein αsubunit sequence, or fragment thereof. In some embodiments, the peptidethat inhibits a binding interaction between an a β integrin and a Gprotein α subunit comprises an amino acid sequence which has at least25% sequence identity to the amino acid sequence of a human wild-type Gprotein α subunit, e.g., SEQ ID NO: 10 or 11 or a fragment thereof(e.g., a fragment of about 4 to about 15 contiguous amino acids of SEQID NO: 10 or 11). In some embodiments, the compound comprises an aminoacid sequence which is at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or has greater than 95% sequence identity toSEQ ID NO: 10 or 11 or a fragment thereof (e.g., a fragment of about 4to about 10 contiguous amino acids of SEQ ID NO: 10 or 11). In someembodiments, the compound comprises an amino acid sequence which is atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, orhas greater than 95% sequence identity to amino acids 201-216 of Gα₁₂(SEQ ID NO: 11) or amino acids 197-212 of Gα₁₃ (SEQ ID NO: 10), aminoacids 197-209 of Gα₁₃ (SEQ ID NO: 10) or amino acids 198-206 of Gα₁₃(SEQ ID NO: 10), amino acids 203-211 of Gα₁₂ (SEQ ID NO: 11). In someembodiments, the compound comprises an amino acid sequence which is atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, orhas greater than 95% sequence identity to SEQ ID NOs: 40.

In exemplary embodiments, the compound comprises an amino acid sequence:Xaa₁ Xaa₂ Glu,wherein Xaa₁ is Glu or Gln and Xaa₂ is Glu, Lys, Ser, or Ala. Inexemplary aspects, (i) Xaa₁ is Glu and Xaa₂ is Glu, Lys, or Ala or (ii)Xaa₁ is Gln and Xaa₂ is Ser. In exemplary aspects, each of Xaa₁ and Xaa₂is Glu. In exemplary aspects, the peptide comprises Phe N-terminal toXaa₁ Xaa₂ Glu. In exemplary aspects, the peptide comprises ArgC-terminal to Xaa₁ Xaa₂ Glu. In exemplary aspects, the peptide comprisesPhe N-terminal to Xaa₁ Xaa₂ Glu and Arg C-terminal to Xaa₁ Xaa₂ Glu. Inexemplary aspects, the compound comprises an extension of two aminoacids, Xaa⁻¹ Xaa₀ N-terminal to Xaa₁ Xaa₂ Glu. In exemplary aspects,Xaa⁻¹¹ of the N-terminal extension is Lys or Arg. In exemplary aspects,Xaa₀ of the N-terminal extension is Phe. In exemplary aspects, thecompound comprises an extension of two amino acids Xaa₃ Xaa₄ C terminalto Xaa₁ Xaa₂ Glu. In exemplary aspects, Xaa₃ of the C-terminal extensionis Lys, Arg, or Gln. In exemplary aspects, Xaa₄ of the C-terminalextension is Met, Leu, Ala, Ser, or Gln. In exemplary aspects, Xaa₃ isArg and Xaa₄ is Ala. Accordingly, in exemplary aspects of the inventionthe compound comprises a sequence selected from the group consisting of:HDRKEFAKFEEERARAKWDT (SEQ ID NO: 83) KFEEERARAKWDT (SEQ ID NO: 22);KFEEERA (SEQ ID NO: 20); and EEERA (SEQ ID NO: 21). In exemplaryaspects, the peptide comprises, consists essentially of, or consists ofFEEERA (SEQ ID NO: 87). In exemplary aspects, the peptide comprises asequence of KFEEE (SEQ ID NO: 19), FEEER (SEQ ID NO: 84), AKFEEE (SEQ IDNO: 85), KFEEER (SEQ ID NO: 86), FEEERA (SEQ ID NO: 87), EEERAR (SEQ IDNO: 88), EEERARA (SEQ ID NO: 89), or EEERARAK (SEQ ID NO: 90).

In exemplary aspects, the peptide comprises a core of EXE (SEQ ID NO:93), wherein X is any amino acid. In exemplary aspects, X is Glu, Lys,Ser, or Ala. In exemplary aspects, X is Ala, Gly, Val, Leu, or Ile. Inexemplary aspects, X is Glu or Asp. In exemplary aspects, X is Ser orThr. In exemplary aspects, X is Lys or omithine or Arg. In exemplaryaspects, X is Pro, Phe, Tyr, Trp, Asn, Gln, His, Cys, or Met.

In exemplary aspects, the peptide comprises FEXE (SEQ ID NO: 94, whereinX is any amino acid. In exemplary aspect, X is Glu or Asp. In exemplaryaspects, X is Ala, Gly, Val, Leu, or Ile. In exemplary aspects, X is Gluor Asp. In exemplary aspects, X is Ser or Thr. In exemplary aspects, Xis Lys or omithine or Arg. In exemplary aspects, X is Pro, Phe, Tyr,Trp, Asn, Gln, His, Cys, or Met.

In exemplary aspects, the peptide comprises FEX₁EX₂ (SEQ ID NO: 414)wherein each of X1 and X2 is independently any amino acid. In exemplaryaspects, the peptide comprises FEX₁EX₂ (SEQ ID NO: 95), wherein X1 isany amino acid and X2 is Lys, Arg, or Gln. In exemplary aspect, X1 isGlu or Asp. In exemplary aspects, X1 is Ala, Gly, Val, Leu, or Ile. Inexemplary aspects, X1 is Glu or Asp. In exemplary aspects, X1 is Ser orThr. In exemplary aspects, X1 is Lys or ornithine or Arg. In exemplaryaspects, X1 is Pro, Phe, Tyr, Trp, Asn, Gln, His, Cys, or Met.

In exemplary aspects, the peptide comprises EX1EX2 (SEQ ID NO: 415)wherein each of X1 and X2 is independently any amino acid. In exemplaryaspects, the peptide comprises EX1EX2 (SEQ ID NO: 96), wherein X1 is anyamino acid and X2 is Lys, Arg, or Gln. In exemplary aspect, X1 is Glu orAsp. In exemplary aspects, X1 is Ala, Gly, Val, Leu, or Ile. Inexemplary aspects, X1 is Glu or Asp. In exemplary aspects, X1 is Ser orThr. In exemplary aspects, X1 is Lys or ornithine or Arg. In exemplaryaspects, X1 is Pro, Phe, Tyr, Trp, Asn, Gln, His, Cys, or Met.

In exemplary aspects, the peptide comprises X1FEX2E (SEQ ID NO: 416)wherein each of X1 and X2 is independently any amino acid. In exemplaryaspects, the peptide comprises X1FEX2E (SEQ ID NO: 97), wherein X1 isLys or Arg and X2 is any amino acid. In exemplary aspect, X2 is Glu orAsp. In exemplary aspects, X2 is Ala, Gly, Val, Leu, or Ile. Inexemplary aspects, X2 is Glu or Asp. In exemplary aspects, X2 is Ser orThr. In exemplary aspects, X2 is Lys or ornithine or Arg. In exemplaryaspects, X2 is Pro, Phe, Tyr, Trp, Asn, Gln, His, Cys, or Met.

In exemplary aspects, the peptide comprises X1FEX2EX3 (SEQ ID NO: 417)wherein each of X1, X2, and X3 is independently any amino acid. Inexemplary aspects, the peptide comprises X1FEX2EX3 (SEQ ID NO: 98),wherein X1 is Lys or Arg, wherein X2 is any amino acid, and X3 is Lys,Arg, or Gln. In exemplary aspect, X2 is Glu or Asp. In exemplaryaspects, X2 is Ala, Gly, Val, Leu, or Ile. In exemplary aspects, X2 isGlu or Asp. In exemplary aspects, X2 is Ser or Thr. In exemplaryaspects, X2 is Lys or ornithine or Arg. In exemplary aspects, X2 is Pro,Phe, Tyr, Trp, Asn, Gln, His, Cys, or Met.

In exemplary aspects, the peptide comprises X1FEX2EX3X4 (SEQ ID NO: 418)wherein each of X1, X2, X3, and X4 is independently any amino acid. Inexemplary aspects, the peptide comprises X1FEX2EX3X4 (SEQ ID NO: 99),wherein X1 is Lys or Arg, X2 is any amino acid, X3 is Lys, Arg, or Gln,and X4 is any amino acid, optionally, Met, Ala, Leu, Ser, or Gln. Inexemplary aspect, X2 is Glu or Asp. In exemplary aspects, X2 is Ala,Gly, Val, Leu, or Ile. In exemplary aspects, X2 is Glu or Asp. Inexemplary aspects, X2 is Ser or Thr. In exemplary aspects, X2 is Lys orornithine or Arg. In exemplary aspects, X2 is Pro, Phe, Tyr, Trp, Asn,Gln, His, Cys, or Met.

In exemplary aspects, the peptide comprises any of SEQ ID NOs: 100-122.

In exemplary embodiments of the invention, the compound comprises theamino acid sequence X₁X₂X₃X₄X₅X₆X₇KX₈X₉X₁₀X₁X₁₂ (SEQ ID NO: 44). Inexemplary aspects, each of X₁, X₂, X₃, X₈, and X₉ is independently analiphatic amino acid. In exemplary aspects, each of X₄, X₅, and X₁₀ isindependently a basic amino acid. In exemplary aspects, X₆ is Pro. Inexemplary aspects, X₇ and X₁₂ is independently a hydroxyl-containingamino acid. In exemplary aspects, X₁₁ is an acidic amino acid. Inexemplary aspects, each of X₁, X₂, X₃, X₈, and X₉ is independentlyselected from the group consisting of L, A, G, and I. In exemplaryaspects, each of X₄, X₅, and X₁₀ is independently selected from thegroup consisting of R and H. In exemplary aspects, X₇ and X₁₂ isindependently a T or Y. In exemplary aspects, X₁₁ is an E or D.Accordingly, in exemplary aspects, the compound comprises the amino acidsequence of LLARRPTKGIHEY (SEQ ID NO: 45).

Additional Peptide Modifications

In alternative or additional embodiments of the invention, the peptideis lipidated (e.g., myritoylated, palmitoylated), glycosylated,amidated, carboxylated, phosphorylated, esterified, acylated,acetylated, cyclized, or converted into an acid addition salt and/oroptionally dimerized or polymerized, or conjugated, as further describedherein.

Lipidation

In exemplary aspects, the peptide is lipidated, or otherwise, attachedto a lipid. The lipid, in some embodiments, is a fatty acid, eicosanoid,prostaglandin, leukotriene, thromboxane, N-acyl ethanolamine),glycerolipid (e.g., mono-, di-, tri-substituted glycerols),glycerophospholipid (e.g., phosphatidylcholine, phosphatidylinositol,phosphatidylethanolamine, phosphatidylserine), sphingolipid (e.g.,sphingosine, ceramide), sterol lipid (e.g., steroid, cholesterol),prenol lipid, saccharolipid, or a polyketide, oil, wax, cholesterol,sterol, fat-soluble vitamin, monoglyceride, diglyceride, triglyceride, aphospholipid.

In exemplary aspects, the peptide is covalently attached to a fattyacid. In some specific embodiments, the fatty acid is a C4 to C30 fattyacid. The fatty acid in exemplary aspects is any of a C4 fatty acid, C6fatty acid, C8 fatty acid, C10 fatty acid, C12 fatty acid, C14 fattyacid, C16 fatty acid, C18 fatty acid, C20 fatty acid, C22 fatty acid,C24 fatty acid, C26 fatty acid, C28 fatty acid, or a C30 fatty acid. Insome embodiments, the fatty acid is a C8 to C20 fatty acid, a C12 to C29fatty acid, or a C14 to C18 fatty acid, e.g., a C14 fatty acid or a C16fatty acid.

In exemplary aspects, the peptide is covalently attached to a fatty acidand the fatty acid is attached to the N-terminal amino acid or theC-terminal amino acid. In alternative aspects, the peptide is covalentlyattached to a fatty acid and the fatty acid is attached to an internalamino acid of the peptide, e.g., via a functional group off of a sidechain of the internal amino acid. For example, the fatty acid may beattached to an amine, hydroxyl, or thiol of a side chain of an internalamino acid. In exemplary aspects, the peptide is covalently attached toa fatty acid and the fatty acid is attached to the second third, fourth,fifth, sixth, seventh, eighth, ninth, tenth, eleventh, or twelfth aminoacid.

In exemplary aspects, the peptide comprises the amino acid sequence ofany one of SEQ ID NOs: 19-39, 84-90, and 93-122 and the peptide iscovalently attached to a C4-C30 fatty acid. In exemplary aspects, thepeptide is any one of SEQ ID NOs: 355-412.

In some aspects, the first amino acid of the peptide is myristoylated atthe N-terminus in which the N-terminal alpha —NH₂ group of an unmodifiedpeptide is attached to a fatty acid. In exemplary aspects, the peptideis any one of SEQ ID NOs: 123-180.

In some aspects, the first amino acid of the peptide is myristoylated atthe N-terminus in which the N-terminal alpha —NH₂ group of an unmodifiedpeptide is attached to a C4-C30 fatty acid. In exemplary aspects, thepeptide is any one of SEQ ID NOs: 181-238.

In some aspects, the first amino acid of the peptide is myristoylated atthe N-terminus in which the N-terminal alpha —NH₂ group of an unmodifiedpeptide is attached to a C12-C18 fatty acid. In exemplary aspects, thepeptide is any one of SEQ ID NOs: 239-296.

In some aspects, the first amino acid of the peptide is myristoylated atthe N-terminus in which the N-terminal alpha —NH₂ group of an unmodifiedpeptide is attached to a myristate. In exemplary aspects, the peptide isany one of SEQ ID NOs: 297-354.

In exemplary aspects, the peptide comprises a my

In exemplary aspects, the lipid attached to the peptide facilitatesmicelle formation of the peptide. For further descriptions of micellarforms of peptides, see the descriptions below under “Micelles.”

Cyclization

In exemplary aspects, the peptide is cyclized. For example, the peptidemay comprise two Cys residues, the sulfur atoms of which participate inthe formation of a disulfide bridge. In exemplary aspects, the peptidecomprises a Cys residue as the terminal residues. Suitable methods ofmodifying peptides with disulfide bridges or sulfur-based cyclizationare described in, for example, Jackson et al., J. Am. Chem. Soc. 113:9391-9392 (1991) and Rudinger and Jost, Experientia 20: 570-571 (1964).

The alpha helix of the analog can alternatively be stabilized throughother means of peptide cyclizing, which means are reviewed in Davies, J.Peptide. Sci. 9: 471-501 (2003). The alpha helix can be stabilized viathe formation of an amide bridge, thioether bridge, thioester bridge,urea bridge, carbamate bridge, sulfonamide bridge, and the like. Forexample, a thioester bridge can be formed between the C-terminus and theside chain of a Cys residue. Alternatively, a thioester can be formedvia side chains of amino acids having a thiol (Cys) and a carboxylicacid (e.g., Asp, Glu). In another method, a cross-linking agent, such asa dicarboxylic acid, e.g., suberic acid (octanedioic acid), etc. canintroduce a link between two functional groups of an amino acid sidechain, such as a free amino, hydroxyl, thiol group, and combinationsthereof.

Peptide Analogs

In some embodiments, the compound is a peptide analog having a structurebased on one of the peptides disclosed herein (the “parent peptide”) butdiffers from the parent peptide in one or more respects. Accordingly, asappreciated by one of ordinary skill in the art the teachings of theparent peptides provided herein may also be applicable the peptideanalogs.

In some aspects, the peptide analog comprises the structure of a parentpeptide, except that the peptide analog comprises one or morenon-peptide bonds in place of peptide bond(s). In exemplary aspects, thepeptide analog comprises in place of a peptide bond, an ester bond, anether bond, a thioether bond, an amide bond, and the like. In someaspects, the peptide analog is a depsipeptide comprising an esterlinkage in place of a peptide bond.

In some aspects, the peptide analog comprises the structure of a parentpeptide described herein, except that the peptide analog comprises oneor more amino acid substitutions, e.g., one or more conservative aminoacid substitutions. Conservative amino acid substitutions are known inthe art, and include amino acid substitutions in which one amino acidhaving certain physical and/or chemical properties is exchanged foranother amino acid that has the same chemical or physical properties.For instance, the conservative amino acid substitution may be an acidicamino acid substituted for another acidic amino acid (e.g., Asp or Glu),an amino acid with a nonpolar side chain substituted for another aminoacid with a nonpolar side chain (e.g., Ala, Gly, Val, Ile, Leu, Met,Phe, Pro, Trp, Val, etc.), a basic amino acid substituted for anotherbasic amino acid (Lys, Arg, etc.), an amino acid with a polar side chainsubstituted for another amino acid with a polar side chain (Asn, Cys,Gln, Ser, Thr, Tyr, etc.), etc.

In some aspects, the peptide analog comprises one or more syntheticamino acids, e.g., an amino acid non-native to a mammal. Synthetic aminoacids include β-alanine (β-Ala), N-α-methyl-alanine (Me-Ala),aminobutyric acid (Abu), γ-aminobutyric acid (γ-Abu), aminohexanoic acid(s-Ahx), aminoisobutyric acid (Aib), aminomethylpyrrole carboxylic acid,aminopiperidinecarboxylic acid, aminoserine (Ams),aminotetrahydropyran-4-carboxylic acid, arginine N-methoxy-N-methylamide, β-aspartic acid (β-Asp), azetidine carboxylic acid,3-(2-benzothiazolyl)alanine, α-tert-butylglycine,2-amino-5-ureido-n-valeric acid (citrulline, Cit), β-Cyclohexylalanine(Cha), acetamidomethyl-cysteine, diaminobutanoic acid (Dab),diaminopropionic acid (Dpr), dihydroxyphenylalanine (DOPA),dimethylthiazolidine (DMTA), γ-Glutamic acid (γ-Glu), homoserine (Hse),hydroxyproline (Hyp), isoleucine N-methoxy-N-methyl amide,methyl-isoleucine (Melle), isonipecotic acid (Isn), methyl-leucine(MeLeu), methyl-lysine, dimethyl-lysine, trimethyl-lysine,methanoproline, methionine-sulfoxide (Met(O)), methionine-sulfone(Met(O₂)), norleucine (Nle), methyl-norleucine (Me-Nle), norvaline(Nva), ornithine (Orn), para-aminobenzoic acid (PABA), penicillamine(Pen), methylphenylalanine (MePhe), 4-Chlorophenylalanine (Phe(4-Cl)),4-fluorophenylalanine (Phe(4-F)), 4-nitrophenylalanine (Phe(4-NO₂)),4-cyanophenylalanine ((Phe(4-CN)), phenylglycine (Phg),piperidinylalanine, piperidinylglycine, 3,4-dehydroproline,pyrrolidinylalanine, sarcosine (Sar), selenocysteine (Sec),O-Benzyl-phosphoserine, 4-amino-3-hydroxy-6-methylheptanoic acid (Sta),4-amino-5-cyclohexyl-3-hydroxypentanoic acid (ACHPA),4-amino-3-hydroxy-5-phenylpentanoic acid (AHPPA),1,2,3,4,-tetrahydro-isoquinoline-3-carboxylic acid (Tic),tetrahydropyranglycine, thienylalanine (Thi), O-benzyl-phosphotyrosine,O-Phosphotyrosine, methoxytyrosine, ethoxytyrosine,O-(bis-dimethylamino-phosphono)-tyrosine, tyrosine sulfatetetrabutylamine, methyl-valine (MeVal), and alkylated3-mercaptopropionic acid.

In some embodiments, the peptide analog comprises one or morenon-conservative amino acid substitutions and the peptide analog stillfunctions to a similar extent, the same extent, or an improved extent asthe parent peptide. In certain aspects, the peptide analog comprisingone or more non-conservative amino acid substitutions inhibits thebinding interaction between β integrin and G protein α subunit to anextent better than the parent peptide.

In some embodiments, and/or one or more amino acid insertions ordeletions, in reference to the parent peptide described herein. In someembodiments, the peptide analog comprises an insertion of one or moreamino acids at the N- or C-terminus in reference to the parent peptide.In some embodiments, the peptide analog comprises a deletion of one ormore amino acids at the N- or C-terminus in reference to the parentpeptide. In these aspects, the peptide analog still functions to asimilar extent, the same extent, or an improved extent as the parentpeptide to inhibit the binding interaction between β integrin and Gprotein α subunit.

In some aspects, the peptide analog is a peptidomimetic. Peptidomimeticsas well as methods of making the same are known in the art. See, forexample, Advances in Amino Acid Mimetics and Peptidomimetics, Volumes 1and 2, ed., Abell, A., JAI Press Inc., Greenwich, C T, 2006. In someaspects, the peptidomimetic is a D-peptide peptidomimetic comprisingD-isomer amino acids. In some aspects, the peptidomimetic is a peptoidin which the side chain of an amino acid is connected to the alphanitrogen atom of the peptide backbone. Methods of making peptoids areknown in the art. See, e.g., Zuckermann et al., JACS 114(26):10646-10647 (1992) and Design, Synthesis, and Evaluation of NovelPeptoids, Fowler, Sarah, University of Wisconsin-Madison, 2008. In someaspects, the peptidomimetic is a β-peptide comprising β amino acidswhich have their amino group bonded to the β-cargon rather than thealpha carbon. Methods of making β-peptides are known in the art. See,for example, Seebach et al., Helvetica Chimica Acta 79(4): 913-941(1996).

Pharmaceutically Acceptable Salts

With regard to the invention, the compounds that inhibit a bindinginteraction between a β integrin and a G protein α subunit, (whichcompounds are collectively referred to hereinafter as “active agents”)in some aspects is in the form of a salt, e.g., a pharmaceuticallyacceptable salt. Such salts can be prepared in situ during the finalisolation and purification of the active agent or separately prepared byreacting a free base function with a suitable acid. Examples of acidswhich can be employed to form pharmaceutically acceptable acid additionsalts include, for example, an inorganic acid, e.g., hydrochloric acid,hydrobromic acid, sulphuric acid, and phosphoric acid, and an organicacid, e.g., oxalic acid, maleic acid, succinic acid, and citric acid.

Representative acid addition salts include, but are not limited toacetate, adipate, alginate, citrate, aspartate, benzoate,benzenesulfonate, bisulfate, butyrate, camphorate, camphor sulfonate,digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate,fumarate, hydrochloride, hydrobromide, hydroiodide,2-hydroxyethansulfonate (isothionate), lactate, maleate, methanesulfonate, nicotinate, 2-naphthalene sulfonate, oxalate, palmitoate,pectinate, persulfate, 3-phenylpropionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, phosphate, glutamate,bicarbonate, p-toluenesulfonate, and undecanoate.

Basic addition salts also can be prepared in situ during the finalisolation and purification of the active agent, or by reacting acarboxylic acid-containing moiety with a suitable base such as thehydroxide, carbonate, or bicarbonate of a pharmaceutically acceptablemetal cation or with ammonia or an organic primary, secondary, ortertiary amine. Pharmaceutically acceptable salts include, but are notlimited to, cations based on alkali metals or alkaline earth metals suchas lithium, sodium, potassium, calcium, magnesium, and aluminum salts,and the like, and nontoxic quaternary ammonia and amine cationsincluding ammonium, tetramethylammonium, tetraethylammonium,methylammonium, dimethylammonium, trimethylammonium, triethylammonium,diethylammonium, and ethylammonium, amongst others. Other representativeorganic amines useful for the formation of base addition salts include,for example, ethylenediamine, ethanolamine, diethanolamine, piperidine,piperazine, and the like.

Further, basic nitrogen-containing groups can be quaternized with suchactive agents as lower alkyl halides such as methyl, ethyl, propyl, andbutyl chlorides, bromides, and iodides; long chain halides such asdecyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides;arylalkyl halides like benzyl and phenethyl bromides and others. Wateror oil-soluble or dispersible products are thereby obtained.

Isolated and Purified

The compounds of the invention that inhibit a binding interactionbetween a β integrin and a G protein α subunit can be isolated and/orpurified. The term “isolated” as used herein means having been removedfrom its natural environment. The term “purified” as used herein meanshaving been increased in purity, wherein “purity” is a relative term,and not to be necessarily construed as absolute purity. In exemplaryaspects, the purity of the compound (e.g., in the composition) is atleast or about 50%, at least or about 60%, at least or about 70%, atleast or about 80%, at least or about 90%, at least or about 95%, or atleast or about 98% or is about 100%.

Methods of Making Peptides

The peptides of the present disclosure may be obtained by methods knownin the art. Suitable methods of de novo synthesizing peptides aredescribed in, for example, Chan et al., Fmoc Solid Phase PeptideSynthesis, Oxford University Press, Oxford, United Kingdom, 2005;Peptide and Protein Drug Analysis, ed. Reid, R., Marcel Dekker, Inc.,2000; Epitope Mapping, ed. Westwood et al., Oxford University Press,Oxford, United Kingdom, 2000; and U.S. Pat. No. 5,449,752. Additionalexemplary methods of making the peptides of the invention are set forthherein.

In some embodiments, the peptides described herein are commerciallysynthesized by companies, such as Synpep (Dublin, Calif.), PeptideTechnologies Corp. (Gaithersburg, Md.), Multiple Peptide Systems (SanDiego, Calif.), Peptide 2.0 Inc. (Chantilly, Va.), and American PeptideCo. (Sunnyvale, Calif.). In this respect, the peptides can be synthetic,recombinant, isolated, and/or purified.

Also, in some aspects, the peptides are recombinantly produced using anucleic acid encoding the amino acid sequence of the peptide usingstandard recombinant methods. See, for instance, Sambrook et al.,Molecular Cloning: A Laboratory Manual. 3rd ed., Cold Spring HarborPress, Cold Spring Harbor, N.Y. 2001; and Ausubel et al., CurrentProtocols in Molecular Biology, Greene Publishing Associates and JohnWiley & Sons, N Y, 1994.

Nucleic Acids

In exemplary embodiments of the invention, the compound that inhibits abinding interaction between a β integrin and a G protein α subunitcomprises or is a nucleic acid comprising a nucleotide sequence encodingany of the antibodies or peptides described herein (including analogsthereof). The nucleic acid can comprise any nucleotide sequence whichencodes any of the antibodies, peptides, or analogs thereof.

In exemplary embodiments of the invention, the compound is a nucleicacid which inhibits expression of the β integrin or the G protein αsubunit. In exemplary aspects, the compound is an antisense molecule, amicroRNA (miRNA), small hairpin (shRNA), and the like. In exemplaryembodiments of the invention, the compound is a nucleic acid whichinhibits expression of any of the β integrins or G protein α subunitsdescribed herein. In exemplary aspects, the compound is a smallinterfering RNA molecule (siRNA). In some aspects, the siRNA inhibitsthe expression Gα₁₃. For example, the siRNA comprises the sequence ofSEQ ID NO: 7 or 8.

By “nucleic acid” as used herein includes “polynucleotide,”“oligonucleotide,” and “nucleic acid molecule,” and generally means apolymer of DNA or RNA, which can be single-stranded or double-stranded,synthesized or obtained (e.g., isolated and/or purified) from naturalsources, which can contain natural, non-natural or altered nucleotides,and which can contain a natural, non-natural or altered inter-nucleotidelinkage, such as a phosphoroamidate linkage or a phosphorothioatelinkage, instead of the phosphodiester found between the nucleotides ofan unmodified oligonucleotide. In some embodiments, the nucleic aciddoes not comprise any insertions, deletions, inversions, and/orsubstitutions. In other embodiments, the nucleic acid comprises one ormore insertions, deletions, inversions, and/or substitutions.

In some aspects, the nucleic acids of the invention are recombinant. Asused herein, the term “recombinant” refers to (i) molecules that areconstructed outside living cells by joining natural or synthetic nucleicacid segments to nucleic acid molecules that can replicate in a livingcell, or (ii) molecules that result from the replication of thosedescribed in (i) above. For purposes herein, the replication can be invitro replication or in vivo replication.

The nucleic acids in some aspects are constructed based on chemicalsynthesis and/or enzymatic ligation reactions using procedures known inthe art. See, for example, Sambrook et al., supra; and Ausubel et al.,supra. For example, a nucleic acid can be chemically synthesized usingnaturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed upon hybridization(e.g., phosphorothioate derivatives and acridine substitutednucleotides). Examples of modified nucleotides that can be used togenerate the nucleic acids include, but are not limited to,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridme,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N-substitutedadenine, 7-methylguanine, 5-methylammomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N⁶-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouratil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleicacids of the invention can be purchased from companies, such asMacromolecular Resources (Fort Collins, Colo.) and Synthegen (Houston,Tex.).

Recombinant Expression Vector

The nucleic acids of the invention in some aspects are incorporated intoa recombinant expression vector. In this regard, the invention providesrecombinant expression vectors comprising any of the presently disclosednucleic acids. For purposes herein, the term “recombinant expressionvector” means a genetically-modified oligonucleotide or polynucleotideconstruct that permits the expression of an mRNA, protein, polypeptide,or peptide by a host cell, when the construct comprises a nucleotidesequence encoding the mRNA, protein, polypeptide, or peptide, and thevector is contacted with the cell under conditions sufficient to havethe mRNA, protein, polypeptide, or peptide expressed within the cell.The vectors of the invention are not naturally-occurring as a whole.However, parts of the vectors can be naturally-occurring. The presentlydisclosed recombinant expression vectors may comprise any type ofnucleotides, including, but not limited to DNA and RNA, which may besingle-stranded or double-stranded, synthesized or obtained in part fromnatural sources, and which can contain natural, non-natural or alterednucleotides. The recombinant expression vectors may comprisenaturally-occurring or non-naturally-occuring internucleotide linkages,or both types of linkages. In some aspects, the altered nucleotides ornon-naturally occurring internucleotide linkages do not hinder thetranscription or replication of the vector.

The recombinant expression vector of the invention can be any suitablerecombinant expression vector, and can be used to transform or transfectany suitable host. Suitable vectors include those designed forpropagation and expansion or for expression or both, such as plasmidsand viruses. The vector can be selected from the group consisting of thepUC series (Fermentas Life Sciences), the pBluescript series(Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison, Wis.),the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series(Clontech, Palo Alto, Calif.). Bacteriophage vectors, such as λGTIO,λGT1 1, λZapII (Stratagene), λEMBL4, and λNM1 149, also can be used.Examples of plant expression vectors include pBIO1, pBI101.2, pBI101.3,pBI121 and pBIN19 (Clontech). Examples of animal expression vectorsinclude pEUK-Cl, pMAM and pMAMneo (Clontech). In some aspects, therecombinant expression vector is a viral vector, e.g., a retroviralvector.

The recombinant expression vectors of the invention can be preparedusing standard recombinant DNA techniques described in, for example,Sambrook et al., supra, and Ausubel et al., supra. Constructs ofexpression vectors, which are circular or linear, can be prepared tocontain a replication system functional in a prokaryotic or eukaryotichost cell. Replication systems can be derived, e.g., from CoIE1, 2μplasmid, λ, SV40, bovine papilloma virus, and the like.

In some aspects, the recombinant expression vector comprises regulatorysequences, such as transcription and translation initiation andtermination codons, which are specific to the type of host (e.g.,bacterium, fungus, plant, or animal) into which the vector is to beintroduced, as appropriate and taking into consideration whether thevector is DNA- or RNA-based.

The recombinant expression vector may include one or more marker genes,which allow for selection of transformed or transfected hosts. Markergenes include biocide resistance, e.g., resistance to antibiotics, heavymetals, etc., complementation in an auxotrophic host to provideprototrophy, and the like. Suitable marker genes for the presentlydisclosed expression vectors include, for instance, neomycin/G418resistance genes, hygromycin resistance genes, histidinol resistancegenes, tetracycline resistance genes, and ampicillin resistance genes.

The recombinant expression vector can comprise a native or normativepromoter operably linked to the nucleotide sequence encoding thepolypeptide (including functional portions and functional variantsthereof), or to the nucleotide sequence which is complementary to orwhich hybridizes to the nucleotide sequence encoding the polypeptide.The selection of promoters, e.g., strong, weak, inducible,tissue-specific and developmental-specific, is within the ordinary skillof the artisan. Similarly, the combining of a nucleotide sequence with apromoter is also within the skill of the artisan. The promoter can be anon-viral promoter or a viral promoter, e.g., a cytomegalovirus (CMV)promoter, an SV40 promoter, an RSV promoter, and a promoter found in thelong-terminal repeat of the murine stem cell virus.

The presently disclosed recombinant expression vectors may be designedfor either transient expression, for stable expression, or for both.Also, the recombinant expression vectors may be made for constitutiveexpression or for inducible expression. Further, the recombinantexpression vectors may be made to include a suicide gene.

As used herein, the term “suicide gene” refers to a gene that causes thecell expressing the suicide gene to die. The suicide gene in someaspects is a gene that confers sensitivity to an agent, e.g., a drug,upon the cell in which the gene is expressed, and causes the cell to diewhen the cell is contacted with or exposed to the agent. Suicide genesare known in the art (see, for example, Suicide Gene Therapy: Methodsand Reviews. Springer, Caroline J. (Cancer Research UK Centre for CancerTherapeutics at the Institute of Cancer Research, Sutton, Surrey, UK),Humana Press, 2004) and include, for example, the Herpes Simplex Virus(HSV) thymidine kinase (TK) gene, cytosine daminase, purine nucleosidephosphorylase, and nitroreductase.

Host Cells

In exemplary embodiments, the compound is a cell expressing the nucleicacid of the invention, optionally, wherein the nucleic acid is pary of arecombinant expression vector. As used herein, the term “host cell”refers to any type of cell that can contain the presently disclosedrecombinant expression vector. The host cell in some aspects is aeukaryotic cell, e.g., plant, animal, fungi, or algae, or can be aprokaryotic cell, e.g., bacteria or protozoa. The host cell in someaspects is a cultured cell or a primary cell, i.e., isolated directlyfrom an organism, e.g., a human. The host cell in some aspects is anadherent cell or a suspended cell, i.e., a cell that grows insuspension. Suitable host cells are known in the art and include, forinstance, DH5α E. coli cells, Chinese hamster ovarian cells, monkey VEROcells, COS cells, HEK293 cells, and the like. For purposes of amplifyingor replicating the recombinant expression vector, the host cell is insome aspects is a prokaryotic cell, e.g., a DH5α cell. For purposes ofproducing a recombinant polypeptide the host cell is in some aspects amammalian cell, e.g., a human cell. The host cell may be of any celltype, can originate from any type of tissue, and can be of anydevelopmental stage.

Also provided by the invention is a population of cells comprising atleast one host cell described herein. The population of cells in someaspects is a heterogeneous population comprising the host cellcomprising any of the recombinant expression vectors described, inaddition to at least one other cell, which does not comprise any of therecombinant expression vectors. Alternatively, in some aspects, thepopulation of cells is a substantially homogeneous population, in whichthe population comprises mainly of host cells (e.g., consistingessentially of) comprising the recombinant expression vector. Thepopulation in some aspects is a clonal population of cells, in which allcells of the population are clones of a single host cell comprising arecombinant expression vector, such that all cells of the populationcomprise the recombinant expression vector. In one embodiment of theinvention, the population of cells is a clonal population comprisinghost cells comprising a recombinant expression vector as describedherein.

Conjugates

In some embodiments, the compounds of the invention are attached orlinked or conjugated to a second moiety (e.g., a heterologous moiety, aconjugate moiety). As used herein, the term “heterologous moiety” issynonomous with “conjugate moiety” and refers to any molecule (chemicalor biochemical, naturally-occurring or non-coded) which is differentfrom the compounds of the invention. Exemplary heterologous moietiesinclude, but are not limited to, a polymer, a carbohydrate, a lipid, anucleic acid, an oligonucleotide, a DNA or RNA, an amino acid, peptide,polypeptide, protein, therapeutic agent, (e.g., a cytotoxic agent,cytokine), or a diagnostic agent.

In some embodiments, the compounds are chemically modified with varioussubstituents. In some embodiments, the chemical modifications impartadditional desirable characteristics as discussed herein. Chemicalmodifications in some aspects take a number of different forms such asheterologous peptides, polysaccarides, lipids, radioisotopes,non-standard amino acid resides and nucleic acids, metal chelates, andvarious cytotoxic agents.

In some embodiments, the compounds are fused to heterologous peptides toconfer various properties, e.g., increased solubility and/or stabilityand/or half-life, resistance to proteolytic cleavage, modulation ofclearance, targeting to particular cell or tissue types. In someembodiments, the compound is linked to a Fc domain of IgG or otherimmunoglobulin. In some embodiments, the compound is fused to alkalinephosphatase (AP). Methods for making Fc or AP fusion constructs arefound in WO 02/060950. By fusing the compound with protein domains thathave specific properties (e.g. half life, bioavailability) it ispossible to confer these properties to the the compound of theinvention.

When the compounds are peptides, they can be modified, for instance, byglycosylation, amidation, carboxylation, or phosphorylation, or by thecreation of acid addition salts, amides, esters, in particularC-terminal esters, and N-acyl derivatives, as discussed above. Thepeptides also can be modified to create peptide derivatives by formingcovalent or noncovalent complexes with other moieties. Covalently boundcomplexes can be prepared by linking the chemical moieties to functionalgroups on the side chains of amino acids comprising the peptides, or atthe N- or C-terminus.

Peptides can be conjugated to a reporter group, including, but notlimited to a radiolabel, a fluorescent label, an enzyme (e.g., thatcatalyzes a calorimetric or fluorometric reaction), a substrate, a solidmatrix, or a carrier (e.g., biotin or avidin). Examples of analogs aredescribed in WO 98/28621 and in Olofsson, et al, Proc. Nat'l. Acad. Sci.USA, 95:11709-11714 (1998), U.S. Pat. Nos. 5,512,545, and 5,474,982;U.S. Patent Application Nos. 20020164687 and 20020164710.

Cysteinyl residues most commonly are reacted with haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carbocyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,α-bromo-.beta.(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol,orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylprocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic orcarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing α-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylissurea; 2,4 pentanedione; and transaminase catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues per se has been studiedextensively, with particular interest in introducing spectral labelsinto tyrosyl residues by reaction with aromatic diazonium compounds ortetranitromethane. Most commonly, N-acetylimidizol and tetranitromethaneare used to form O-acetyl tyrosyl species and 3-nitro derivatives,respectively. Tyrosyl residues are iodinated using ¹²⁵I Or ¹³¹I toprepare labeled proteins for use in radioimmunoassay.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R1) such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or 1-ethyl-3 (4azonia 4,4-dimethylpentyl)carbodiimide. Furthermore, aspartyl andglutamyl residues are converted to asparaginyl and glutaminyl residuesby reaction with ammonium ions.

Derivatization with bifunctional agents is useful for crosslinking thebinding construct to water-insoluble support matrixes. Such derivationmay also provide the linker that may connect adjacent binding elementsin a binding construct, or a binding elements to a heterologous peptide,e.g., a Fc fragment. Commonly used crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homo-bifunctional imidoesters, including disuccinimidyl esterssuch as 3,3′-dithiiobis(succinimidylpropioonate), and bifunctionalmaleimides such as bis-N-maleimido-1,8-octane. Derivatizing agents suchas methyl-3-[(p-azidophenyl) dithio]propioimidate yield photoactivatableintermediates that are capable of forming cross links in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440, incorporated herein by reference, are employed forprotein immobilization.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MoleculeProperties, W. H. Freeman & Co., San Francisco, pp. 79-86,1983),acetylation of the N-terminal amine, and, in some instances, amidationof the C-terminal carboxyl groups. Such derivatives are chemicallymodified polypeptide compositions in which the binding constructpolypeptide is linked to a polymer.

In general, chemical derivatization may be performed under any suitablecondition used to react a protein with an activated polymer molecule.Methods for preparing chemical derivatives of polypeptides willgenerally comprise the steps of (a) reacting the polypeptide with theactivated polymer molecule (such as a reactive ester or aldehydederivative of the polymer molecule) under conditions whereby the bindingconstruct becomes attached to one or more polymer molecules, and (b)obtaining the reaction product(s). The optimal reaction conditions willbe determined based on known parameters and the desired result. Forexample, the larger the ratio of polymer molecules:protein, the greaterthe amount of attached polymer molecule. In some embodiments, thecompound may have a single polymer molecule moiety at the aminoterminus. (See, e.g., U.S. Pat. No. 5,234,784).

Derivatized binding constructs disclosed herein may have additionalactivities, enhanced or reduced biological activity, or othercharacteristics, such as increased or decreased half-life, as comparedto the non-derivatized molecules.

In some embodiments, the compound is directly joined to a conjugatemoiety in the absence of a linker. In alternative aspects, the compoundis indirectly connected to the conjugate moiety via one or more linkers.Whether directly joined together or indirectly joined together through alinker, the compound may be connected through covalent bonds (e.g., apeptide, ester, amide, or sulfhydryl bond) or non-covalent bonds (e.g.,via hydrophobic interaction, hydrogen bond, van der Waals bond,electrostatic or ionic interaction), or a combination thereof. Thecompound of the invention and conjugate moiety may be connected via anymeans known in the art, including, but not limited to, via a linker ofany of the invention. See, for example, the section herein entitled“Linkers.”

Conjugates: Fc Fusions

For substituents such as an Fc region of human IgG, the fusion can befused directly to a compound of the invention or fused through anintervening sequence. For example, a human IgG hinge, CH2 and CH3 regionmay be fused at either the N-terminus or C-terminus of a bindingconstruct to attach the Fc region. The resulting Fc-fusion constructenables purification via a Protein A affinity column (Pierce, Rockford,Ill.). Peptide and proteins fused to an Fc region can exhibit asubstantially greater half-life in vivo than the unfused counterpart. Afusion to an Fc region allows for dimerization/multimerization of thefusion polypeptide. The Fc region may be a naturally occurring Fcregion, or may be modified for superior characteristics, e.g.,therapeutic qualities, circulation time, reduced aggregation. As notedabove, in some embodiments, the compounds are conjugated, e.g., fused toan immunoglobulin or portion thereof (e.g., variable region, CDR, or Fcregion). Known types of immunoglobulins (Ig) include IgG, IgA, IgE, IgDor IgM. The Fc region is a C-terminal region of an Ig heavy chain, whichis responsible for binding to Fc receptors that carry out activitiessuch as recycling (which results in prolonged half-life), antibodydependent cell-mediated cytotoxicity (ADCC), and complement dependentcytotoxicity (CDC).

For example, according to some definitions the human IgG heavy chain Fcregion stretches from Cys226 to the C-terminus of the heavy chain. The“hinge region” generally extends from Glu216 to Pro230 of human IgG1(hinge regions of other IgG isotypes may be aligned with the IgG1sequence by aligning the cysteines involved in cysteine bonding). The Fcregion of an IgG includes two constant domains, CH2 and CH3. The CH2domain of a human IgG Fc region usually extends from amino acids 231 toamino acid 341. The CH3 domain of a human IgG Fc region usually extendsfrom amino acids 342 to 447. References made to amino acid numbering ofimmunoglobulins or immunoglobulin fragments, or regions, are all basedon Kabat et al. 1991, Sequences of Proteins of Immunological Interest,U.S. Department of Public Health, Bethesda, Md. In a relatedembodiments, the Fc region may comprise one or more native or modifiedconstant regions from an immunoglobulin heavy chain, other than CH1, forexample, the CH2 and CH3 regions of IgG and IgA, or the CH3 and CH4regions of IgE.

Suitable conjugate moieties include portions of immunoglobulin sequencethat include the FcRn binding site. FcRn, a salvage receptor, isresponsible for recycling immunoglobulins and returning them tocirculation in blood. The region of the Fc portion of IgG that binds tothe FcRn receptor has been described based on X-ray crystallography(Burmeister et al. 1994, Nature 372:379). The major contact area of theFc with the FcRn is near the junction of the CH2 and CH3 domains.Fc-FcRn contacts are all within a single Ig heavy chain. The majorcontact sites include amino acid residues 248, 250-257, 272, 285, 288,290-291, 308-311, and 314 of the CH2 domain and amino acid residues385-387, 428, and 433-436 of the CH3 domain.

Some conjugate moieties may or may not include FcγR binding site(s).FcγR are responsible for ADCC and CDC. Examples of positions within theFc region that make a direct contact with FcγR are amino acids 234-239(lower hinge region), amino acids 265-269 (B/C loop), amino acids297-299 (C′/E loop), and amino acids 327-332 (F/G) loop (Sondermann etal., Nature 406: 267-273, 2000). The lower hinge region of IgE has alsobeen implicated in the FcRI binding (Henry, et al., Biochemistry 36,15568-15578, 1997). Residues involved in IgA receptor binding aredescribed in Lewis et al., (J Immunol. 175:6694-701, 2005). Amino acidresidues involved in IgE receptor binding are described in Sayers et al.(J Biol Chem. 279(34):35320-5, 2004).

Amino acid modifications may be made to the Fc region of animmunoglobulin. Such variant Fc regions comprise at least one amino acidmodification in the CH3 domain of the Fc region (residues 342-447)and/or at least one amino acid modification in the CH2 domain of the Fcregion (residues 231-341). Mutations believed to impart an increasedaffinity for FcRn include T256A, T307A, E380A, and N434A (Shields et al.2001, J. Biol. Chem. 276:6591). Other mutations may reduce binding ofthe Fc region to FcγRI, FcγRIIA, FcγRIIB, and/or FcγRIIIA withoutsignificantly reducing affinity for FcRn. For example, substitution ofthe Asn at position 297 of the Fc region with Ala or another amino acidremoves a highly conserved N-glycosylation site and may result inreduced immunogenicity with concomitant prolonged half-life of the Fcregion, as well as reduced binding to FcγRs (Routledge et al. 1995,Transplantation 60:847; Friend et al. 1999, Transplantation 68:1632;Shields et al. 1995, J. Biol. Chem. 276:6591). Amino acid modificationsat positions 233-236 of IgG1 have been made that reduce binding to FcγRs(Ward and Ghetie 1995, Therapeutic Immunology 2:77 and Armour et al.1999, Eur. J. Immunol. 29:2613). Some exemplary amino acid substitutionsare described in U.S. Pat. Nos. 7,355,008 and 7,381,408, eachincorporated by reference herein in its entirety.

Heterologous Moieties: Polymers, Carbohydrates, and Lipids

In some embodiments, the heterologous moiety is a polymer. The polymermay be branched or unbranched. The polymer may be of any molecularweight. The polymer in some embodiments has an average molecular weightof between about 2 kDa to about 100 kDa (the term “about” indicatingthat in preparations of a water soluble polymer, some molecules willweigh more, some less, than the stated molecular weight). The averagemolecular weight of the polymer is in some aspect between about 5 kDaand about 50 kDa, between about 12 kDa to about 40 kDa or between about20 kDa to about 35 kDa.

In some embodiments, the polymer is modified to have a single reactivegroup, such as an active ester for acylation or an aldehyde foralkylation, so that the degree of polymerization may be controlled. Thepolymer in some embodiments is water soluble so that the protein towhich it is attached does not precipitate in an aqueous environment,such as a physiological environment. In some embodiments, when, forexample, the composition is used for therapeutic use, the polymer ispharmaceutically acceptable. Additionally, in some aspects, the polymeris a mixture of polymers, e.g., a co-polymer, a block co-polymer.

In some embodiments, the polymer is selected from the group consistingof: polyamides, polycarbonates, polyalkylenes and derivatives thereofincluding, polyalkylene glycols, polyalkylene oxides, polyalkyleneterepthalates, polymers of acrylic and methacrylic esters, includingpoly(methyl methacrylate), poly(ethyl methacrylate),poly(butylmethacrylate), poly(isobutyl methacrylate),poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(laurylmethacrylate), poly(phenyl methacrylate), poly(methyl acrylate),poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecylacrylate), polyvinyl polymers including polyvinyl alcohols, polyvinylethers, polyvinyl esters, polyvinyl halides, poly(vinyl acetate), andpolyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes andco-polymers thereof, celluloses including alkyl cellulose, hydroxyalkylcelluloses, cellulose ethers, cellulose esters, nitro celluloses, methylcellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propylmethyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate,cellulose propionate, cellulose acetate butyrate, cellulose acetatephthalate, carboxylethyl cellulose, cellulose triacetate, and cellulosesulphate sodium salt, polypropylene, polyethylenes includingpoly(ethylene glycol), poly(ethylene oxide), and poly(ethyleneterephthalate), and polystyrene.

In some aspects, the polymer is a biodegradable polymer, including asynthetic biodegradable polymer (e.g., polymers of lactic acid andglycolic acid, polyanhydrides, poly(ortho)esters, polyurethanes,poly(butic acid), poly(valeric acid), and poly(lactide-cocaprolactone)),and a natural biodegradable polymer (e.g., alginate and otherpolysaccharides including dextran and cellulose, collagen, chemicalderivatives thereof (substitutions, additions of chemical groups, forexample, alkyl, alkylene, hydroxylations, oxidations, and othermodifications routinely made by those skilled in the art), albumin andother hydrophilic proteins (e.g., zein and other prolamines andhydrophobic proteins)), as well as any copolymer or mixture thereof. Ingeneral, these materials degrade either by enzymatic hydrolysis orexposure to water in vivo, by surface or bulk erosion.

In some aspects, the polymer is a bioadhesive polymer, such as abioerodible hydrogel described by H. S. Sawhney, C. P. Pathak and J. A.Hubbell in Macromolecules, 1993, 26, 581-587, the teachings of which areincorporated herein, polyhyaluronic acids, casein, gelatin, glutin,polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methylmethacrylates), poly(ethyl methacrylates), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), and poly(octadecyl acrylate).

In some embodiments, the polymer is a water-soluble polymer or ahydrophilic polymer. Suitable water-soluble polymers are known in theart and include, for example, polyvinylpyrrolidone, hydroxypropylcellulose (HPC; Klucel), hydroxypropyl methylcellulose (HIPMC;Methocel), nitrocellulose, hydroxypropyl ethylcellulose, hydroxypropylbutylcellulose, hydroxypropyl pentylcellulose, methyl cellulose,ethylcellulose (Ethocel), hydroxyethyl cellulose, various alkylcelluloses and hydroxyalkyl celluloses, various cellulose ethers,cellulose acetate, carboxymethyl cellulose, sodium carboxymethylcellulose, calcium carboxymethyl cellulose, vinyl acetate/crotonic acidcopolymers, poly-hydroxyalkyl methacrylate, hydroxymethyl methacrylate,methacrylic acid copolymers, polymethacrylic acid,polymethylmethacrylate, maleic anhydride/methyl vinyl ether copolymers,poly vinyl alcohol, sodium and calcium polyacrylic acid, polyacrylicacid, acidic carboxy polymers, carboxypolymethylene, carboxyvinylpolymers, polyoxyethylene polyoxypropylene copolymer,polymethylvinylether co-maleic anhydride, carboxymethylamide, potassiummethacrylate divinylbenzene co-polymer, polyoxyethyleneglycols,polyethylene oxide, and derivatives, salts, and combinations thereof. Insome aspects, the water soluble polymers or mixtures thereof include,but are not limited to, N-linked or O-linked carbohydrates, sugars,phosphates, carbohydrates; sugars; phosphates; polyethylene glycol (PEG)(including the forms of PEG that have been used to derivatize proteins,including mono-(C1-C10) alkoxy- or aryloxy-polyethylene glycol);monomethoxy-polyethylene glycol; dextran (such as low molecular weightdextran, of, for example about 6 kD), cellulose; cellulose; othercarbohydrate-based polymers, poly-(N-vinyl pyrrolidone)polyethyleneglycol, propylene glycol homopolymers, a polypropylene oxide/ethyleneoxide co-polymer, polyoxyethylated polyols (e.g., glycerol) andpolyvinyl alcohol. Also encompassed by the present invention arebifunctional crosslinking molecules which may be used to preparecovalently attached multimers.

A particularly preferred water-soluble polymer for use herein ispolyethylene glycol (PEG). As used herein, polyethylene glycol is meantto encompass any of the forms of PEG that can be used to derivatizeother proteins, such as mono-(C1-C10) alkoxy- or aryloxy-polyethyleneglycol. PEG is a linear or branched neutral polyether, available in abroad range of molecular weights, and is soluble in water and mostorganic solvents. PEG is effective at excluding other polymers orpeptides when present in water, primarily through its high dynamic chainmobility and hydrophibic nature, thus creating a water shell orhydration sphere when attached to other proteins or polymer surfaces.PEG is nontoxic, non-immunogenic, and approved by the Food and DrugAdministration for internal consumption.

Proteins or enzymes when conjugated to PEG have demonstratedbioactivity, non-antigenic properties, and decreased clearance rateswhen administered in animals. F. M. Veronese et al., Preparation andProperties of Monomethoxypoly(ethylene glycol)-modified Enzymes forTherapeutic Applications, in J. M. Harris ed., Poly(Ethylene Glycol)Chemistry—Biotechnical and Biomedical Applications, 127-36, 1992,incorporated herein by reference. These phenomena are due to theexclusion properties of PEG in preventing recognition by the immunesystem. In addition, PEG has been widely used in surface modificationprocedures to decrease protein adsorption and improve bloodcompatibility. S. W. Kim et al., Ann. N.Y. Acad. Sci. 516: 116-30 1987;Jacobs et al., Artif. Organs 12: 500-501, 1988; Park et al., J. Poly.Sci, Part A 29:1725-31, 1991, incorporated herein by reference.Hydrophobic polymer surfaces, such as polyurethanes and polystyrene canbe modified by the grafting of PEG (MW 3,400) and employed asnonthrombogenic surfaces. Surface properties (contact angle) can be moreconsistent with hydrophilic surfaces, due to the hydrating effect ofPEG. More importantly, protein (albumin and other plasma proteins)adsorption can be greatly reduced, resulting from the high chainmotility, hydration sphere, and protein exclusion properties of PEG.

PEG (MW 3,400) was determined as an optimal size in surfaceimmobilization studies, Park et al., J. Biomed. Mat. Res. 26:739-45,1992, while PEG (MW 5,000) was most beneficial in decreasing proteinantigenicity. (F. M. Veronese et al., In J. M. Harris, et al.,Poly(Ethylene Glycol) Chemistry—Biotechnical and BiomedicalApplications, 127-36.)

Methods for preparing pegylated compounds may comprise the steps of (a)reacting the compound with polyethylene glycol (such as a reactive esteror aldehyde derivative of PEG) under conditions whereby the compoundbecomes attached to one or more PEG groups, and (b) obtaining thereaction product(s). In general, the optimal reaction conditions for theacylation reactions will be determined based on known parameters and thedesired result. For example, the larger the ratio of PEG: compound, thegreater the percentage of poly-pegylated product. In some embodiments,the compound will have a single PEG moiety at the N-terminus. See U.S.Pat. No. 8,234,784, herein incorporated by reference.

In some embodiments, the heterologous moiety is a carbohydrate. In someembodiments, the carbohydrate is a monosaccharide (e.g., glucose,galactose, fructose), a disaccharide (e.g., sucrose, lactose, maltose),an oligosaccharide (e.g., raffinose, stachyose), a polysaccharide (astarch, amylase, amylopectin, cellulose, chitin, callose, laminarin,xylan, mannan, fucoidan, galactomannan.

In some embodiments, the heterologous moiety is a lipid. The lipid, insome embodiments, is a fatty acid, eicosanoid, prostaglandin,leukotriene, thromboxane, N-acyl ethanolamine), glycerolipid (e.g.,mono-, di-, tri-substituted glycerols), glycerophospholipid (e.g.,phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine,phosphatidylserine), sphingolipid (e.g., sphingosine, ceramide), sterollipid (e.g., steroid, cholesterol), prenol lipid, saccharolipid, or apolyketide, oil, wax, cholesterol, sterol, fat-soluble vitamin,monoglyceride, diglyceride, triglyceride, a phospholipid.

Heterologous Moieties: Therapeutic Agents

In some embodiments, the heterologous moiety is a therapeutic agent. Thetherapeutic agent may be any of those known in the art. Examples oftherapeutic agents that are contemplated herein include, but are notlimited to, natural enzymes, proteins derived from natural sources,recombinant proteins, natural peptides, synthetic peptides, cyclicpeptides, antibodies, receptor agonists, cytotoxic agents,immunoglobins, beta-adrenergic blocking agents, calcium channelblockers, coronary vasodilators, cardiac glycosides, antiarrhythmics,cardiac sympathomemetics, angiotensin converting enzyme (ACE)inhibitors, diuretics, inotropes, cholesterol and triglyceride reducers,bile acid sequestrants, fibrates, 3-hydroxy-3-methylgluteryl (HMG)-CoAreductase inhibitors, niacin derivatives, antiadrenergic agents,alpha-adrenergic blocking agents, centrally acting antiadrenergicagents, vasodilators, potassium-sparing agents, thiazides and relatedagents, angiotensin II receptor antagonists, peripheral vasodilators,antiandrogens, estrogens, antibiotics, retinoids, insulins and analogs,alpha-glucosidase inhibitors, biguanides, meglitinides, sulfonylureas,thizaolidinediones, androgens, progestogens, bone metabolism regulators,anterior pituitary hormones, hypothalamic hormones, posterior pituitaryhormones, gonadotropins, gonadotropin-releasing hormone antagonists,ovulation stimulants, selective estrogen receptor modulators,antithyroid agents, thyroid hormones, bulk forming agents, laxatives,antiperistaltics, flora modifiers, intestinal adsorbents, intestinalanti-infectives, antianorexic, anticachexic, antibulimics, appetitesuppressants, antiobesity agents, antacids, upper gastrointestinal tractagents, anticholinergic agents, aminosalicylic acid derivatives,biological response modifiers, corticosteroids, antispasmodics, 5-HT₄partial agonists, antihistamines, cannabinoids, dopamine antagonists,serotonin antagonists, cytoprotectives, histamine H2-receptorantagonists, mucosal protective agent, proton pump inhibitors, H. pylorieradication therapy, erythropoieses stimulants, hematopoietic agents,anemia agents, heparins, antifibrinolytics, hemostatics, bloodcoagulation factors, adenosine diphosphate inhibitors, glycoproteinreceptor inhibitors, fibrinogen-platelet binding inhibitors,thromboxane-A₂ inhibitors, plasminogen activators, antithromboticagents, glucocorticoids, mineralcorticoids, corticosteroids, selectiveimmunosuppressive agents, antifungals, drugs involved in prophylactictherapy, AIDS-associated infections, cytomegalovirus, non-nucleosidereverse transcriptase inhibitors, nucleoside analog reverse transcriptseinhibitors, protease inhibitors, anemia, Kaposi's sarcoma,aminoglycosides, carbapenems, cephalosporins, glycopoptides,lincosamides, macrolies, oxazolidinones, penicillins, streptogramins,sulfonamides, trimethoprim and derivatives, tetracyclines,anthelmintics, amebicies, biguanides, cinchona alkaloids, folic acidantagonists, quinoline derivatives, Pneumocystis carinii therapy,hydrazides, imidazoles, triazoles, nitroimidzaoles, cyclic amines,neuraminidase inhibitors, nucleosides, phosphate binders, cholinesteraseinhibitors, adjunctive therapy, barbiturates and derivatives,benzodiazepines, gamma aminobutyric acid derivatives, hydantoinderivatives, iminostilbene derivatives, succinimide derivatives,anticonvulsants, ergot alkaloids, antimigrane preparations, biologicalresponse modifiers, carbamic acid eaters, tricyclic derivatives,depolarizing agents, nondepolarizing agents, neuromuscular paralyticagents, CNS stimulants, dopaminergic reagents, monoamine oxidaseinhibitors, COMT inhibitors, alkyl sulphonates, ethylenimines,imidazotetrazines, nitrogen mustard analogs, nitrosoureas,platinum-containing compounds, antimetabolites, purine analogs,pyrimidine analogs, urea derivatives, antracyclines, actinomycinds,camptothecin derivatives, epipodophyllotoxins, taxanes, vinca alkaloidsand analogs, antiandrogens, antiestrogens, nonsteroidal aromataseinhibitors, protein kinase inhibitor antineoplastics,azaspirodecanedione derivatives, anxiolytics, stimulants, monoamindreuptake inhibitors, selective serotonin reuptake inhibitors,antidepressants, benzisooxazole derivatives, butyrophenone derivatives,dibenzodiazepine derivatives, dibenzothiazepine derivatives,diphenylbutylpiperidine derivatives, phenothiazines,thienobenzodiazepine derivatives, thioxanthene derivatives, allergenicextracts, nonsteroidal agents, leukotriene receptor antagonists,xanthines, endothelin receptor antagonist, prostaglandins, lungsurfactants, mucolytics, antimitotics, uricosurics, xanthine oxidaseinhibitors, phosphodiesterase inhibitors, metheamine salts, nitrofuranderivatives, quinolones, smooth muscle relaxants, parasympathomimeticagents, halogenated hydrocarbons, esters of amino benzoic acid, amides(e.g. lidocaine, articaine hydrochloride, bupivacaine hydrochloride),antipyretics, hynotics and sedatives, cyclopyrrolones,pyrazolopyrimidines, nonsteroidal anti-inflammatory drugs, opioids,para-aminophenol derivatives, alcohol dehydrogenase inhibitor, heparinantagonists, adsorbents, emetics, opoid antagonists, cholinesterasereactivators, nicotine replacement therapy, vitamin A analogs andantagonists, vitamin B analogs and antagonists, vitamin C analogs andantagonists, vitamin D analogs and antagonists, vitamin E analogs andantagonists, vitamin K analogs and antagonists.

The compounds of the invention may be conjugated to one or morecytokines and growth factors that are effective in inhibiting tumormetastasis, and wherein the cytokine or growth factor has been shown tohave an antiproliferative effect on at least one cell population. Suchcytokines, lymphokines, growth factors, or other hematopoietic factorsinclude, but are not limited to: M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14,IL-15, IL-16, IL-17, IL-18, IFN, TNFα, TNF1, TNF2, G-CSF, Meg-CSF,GM-CSF, thrombopoietin, stem cell factor, and erythropoietin. Additionalgrowth factors for use herein include angiogenin, bone morphogenicprotein-1, bone morphogenic protein-2, bone morphogenic protein-3, bonemorphogenic protein-4, bone morphogenic protein-5, bone morphogenicprotein-6, bone morphogenic protein-7, bone morphogenic protein-8, bonemorphogenic protein-9, bone morphogenic protein-10, bone morphogenicprotein-11, bone morphogenic protein-12, bone morphogenic protein-13,bone morphogenic protein-14, bone morphogenic protein-15, bonemorphogenic protein receptor IA, bone morphogenic protein receptor IB,brain derived neurotrophic factor, ciliary neutrophic factor, ciliaryneutrophic factor receptor α, cytokine-induced neutrophil chemotacticfactor 1, cytokine-induced neutrophil, chemotactic factor 2 α,cytokine-induced neutrophil chemotactic factor 2 β, β endothelial cellgrowth factor, endothelin 1, epithelial-derived neutrophil attractant,glial cell line-derived neutrophic factor receptor α 1, glial cellline-derived neutrophic factor receptor α 2, growth related protein,growth related protein α, growth related protein β, growth relatedprotein γ, heparin binding epidermal growth factor, hepatocyte growthfactor, hepatocyte growth factor receptor, insulin-like growth factor I,insulin-like growth factor receptor, insulin-like growth factor II,insulin-like growth factor binding protein, keratinocyte growth factor,leukemia inhibitory factor, leukemia inhibitory factor receptor a, nervegrowth factor nerve growth factor receptor, neurotrophin-3,neurotrophin-4, pre-B cell growth stimulating factor, stem cell factor,stem cell factor receptor, transforming growth factor α, transforminggrowth factor β, transforming growth factor β1, transforming growthfactor β1.2, transforming growth factor β2, transforming growth factorβ3, transforming growth factor β5, latent transforming growth factor β1,transforming growth factor β binding protein I, transforming growthfactor β binding protein II, transforming growth factor β bindingprotein III, tumor necrosis factor receptor type I, tumor necrosisfactor receptor type II, urokinase-type plasminogen activator receptor,and chimeric proteins and biologically or immunologically activefragments thereof.

In some embodiments, the conjugate comprises a compound as describedherein and a cytotoxic agent. The cytotoxic agent is any molecule(chemical or biochemical) which is toxic to a cell. In some aspects,when a cytotoxic agent is conjugated to a compound of the invention, theresults obtained are synergistic. That is to say, the effectiveness ofthe combination therapy of a compound and the cytotoxic agent issynergistic, i.e., the effectiveness is greater than the effectivenessexpected from the additive individual effects of each. Therefore, thedosage of the cytotoxic agent can be reduced and thus, the risk of thetoxicity problems and other side effects is concomitantly reduced. Insome embodiments, the cytotoxic agent is a chemotherapeutic agent.Chemotherapeutic agents are known in the art and include, but notlimited to, platinum coordination compounds, topoisomerase inhibitors,antibiotics, antimitotic alkaloids and difluoronucleosides, as describedin U.S. Pat. No. 6,630,124.

In some embodiments, the chemotherapeutic agent is a platinumcoordination compound. The term “platinum coordination compound” refersto any tumor cell growth inhibiting platinum coordination compound thatprovides the platinum in the form of an ion. In some embodiments, theplatinum coordination compound is cis-diamminediaquoplatinum (II)-ion;chloro(diethylenetriamine)-platinum(II)chloride;dichloro(ethylenediamine)-platinum(II),diammine(1,1-cyclobutanedicarboxylato) platinum(II) (carboplatin);spiroplatin; iproplatin; diammine(2-ethylmalonato)-platinum(II);ethylenediaminemalonatoplatinum(II);aqua(1,2-diaminodyclohexane)-sulfatoplatinum(II);(1,2-diaminocyclohexane)malonatoplatinum(II);(4-caroxyphthalato)(1,2-diaminocyclohexane)platinum(II);(1,2-diaminocyclohexane)-(isocitrato)platinum(II);(1,2-diaminocyclohexane)cis(pyruvato)platinum(II);(1,2-diaminocyclohexane)oxalatoplatinum(II); ormaplatin; andtetraplatin.

In some embodiments, cisplatin is the platinum coordination compoundemployed in the compositions and methods of the present invention.Cisplatin is commercially available under the name PLATINOL™ fromBristol Myers-Squibb Corporation and is available as a powder forconstitution with water, sterile saline or other suitable vehicle. Otherplatinum coordination compounds suitable for use in the presentinvention are known and are available commercially and/or can beprepared by conventional techniques. Cisplatin, orcis-dichlorodiammineplatinum II, has been used successfully for manyyears as a chemotherapeutic agent in the treatment of various humansolid malignant tumors. More recently, other diamino-platinum complexeshave also shown efficacy as chemotherapeutic agents in the treatment ofvarious human solid malignant tumors. Such diamino-platinum complexesinclude, but are not limited to, spiroplatinum and carboplatinum.Although cisplatin and other diamino-platinum complexes have been widelyused as chemotherapeutic agents in humans, they have had to be deliveredat high dosage levels that can lead to toxicity problems such as kidneydamage.

In some embodiments, the chemotherapeutic agent is a topoisomeraseinhibitor. Topoisomerases are enzymes that are capable of altering DNAtopology in eukaryotic cells. They are critical for cellular functionsand cell proliferation. Generally, there are two classes oftopoisomerases in eukaryotic cells, type I and type II. Topoisomerase Iis a monomeric enzyme of approximately 100,000 molecular weight. Theenzyme binds to DNA and introduces a transient single-strand break,unwinds the double helix (or allows it to unwind), and subsequentlyreseals the break before dissociating from the DNA strand. Varioustopoisomerase inhibitors have recently shown clinical efficacy in thetreatment of humans afflicted with ovarian, cancer, esophageal cancer ornon-small cell lung carcinoma.

In some aspects, the topoisomerase inhibitor is camptothecin or acamptothecin analog. Camptothecin is a water-insoluble, cytotoxicalkaloid produced by Camptotheca accuminata trees indigenous to Chinaand Nothapodytes foetida trees indigenous to India. Camptothecinexhibits tumor cell growth inhibiting activity against a number of tumorcells. Compounds of the camptothecin analog class are typically specificinhibitors of DNA topoisomerase I. By the term “inhibitor oftopoisomerase” is meant any tumor cell growth inhibiting compound thatis structurally related to camptothecin. Compounds of the camptothecinanalog class include, but are not limited to; topotecan, irinotecan and9-aminocamptothecin.

In additional embodiments, the cytotoxic agent is any tumor cell growthinhibiting camptothecin analog claimed or described in: U.S. Pat. No.5,004,758, issued on Apr. 2, 1991 and European Patent Application Number88311366.4, published on Jun. 21, 1989 as 20′ Publication Number EP 0321 122; U.S. Pat. No. 4,604,463, issued on Aug. 5, 1986 and EuropeanPatent Application Publication Number EP 0 137 145, published on Apr.17, 1985; U.S. Pat. No. 4,473,692, issued on Sep. 25, 1984 and EuropeanPatent Application Publication Number EP 0 074 256, published on Mar.16, 1983; U.S. Pat. No. 4,545,880, issued on Oct. 8, 1985 and EuropeanPatent Application Publication Number EP 0 074 256, published on Mar.16, 1983; European Patent Application Publication Number EP 0 088 642,published on Sep. 14, 1983; Wani et al., J. Med. Chem., 29, 2358-2363(1986); Nitta et al., Proc. 14th International Congr. Chemotherapy,Kyoto, 1985, Tokyo Press, Anticancer Section 1, p. 28-30, especially acompound called CPT-11. CPT-11 is a camptothecin analog with a4-(piperidino)-piperidine side chain joined through a carbamate linkageat C-10 of 10-hydroxy-7-ethyl camptothecin. CPT-11 is currentlyundergoing human clinical trials and is also referred to as irinotecan;Wani et al, J. Med. Chem., 23, 554 (1980); Wani et. al., J. Med. Chem.,30, 1774 (1987); U.S. Pat. No. 4,342,776, issued on Aug. 3, 1982; U.S.patent application Ser. No. 581,916, filed on Sep. 13, 1990 and EuropeanPatent Application Publication Number EP 418 099, published on Mar. 20,1991; U.S. Pat. No. 4,513,138, issued on Apr. 23, 1985 and EuropeanPatent Application Publication Number EP 0 074 770, published on Mar.23, 1983; U.S. Pat. No. 4,399,276, issued on Aug. 16, 1983 and EuropeanPatent Application Publication Number 0 056 692, published on Jul. 28,1982; the entire disclosure of each of which is hereby incorporated byreference. All of the above-listed compounds of the camptothecin analogclass are available commercially and/or can be prepared by conventionaltechniques including those described in the above-listed references. Thetopoisomerase inhibitor may be selected from the group consisting oftopotecan, irinotecan and 9-aminocamptothecin.

The preparation of numerous compounds of the camptothecin analog class(including pharmaceutically acceptable salts, hydrates and solvatesthereof) as well as the preparation of oral and parenteralpharmaceutical compositions comprising such a compounds of thecamptothecin analog class and an inert, pharmaceutically acceptablecarrier or diluent, is extensively described in U.S. Pat. No. 5,004,758,issued on Apr. 2, 1991 and European Patent Application Number88311366.4, published on Jun. 21, 1989 as Publication Number EP 0 321122, the teachings of which are incorporated herein by reference.

In still yet other embodiments of the invention, the chemotherapeuticagent is an antibiotic compound. Suitable antibiotic include, but arenot limited to, doxorubicin, mitomycin, bleomycin, daunorubicin andstreptozocin.

In some embodiments, the chemotherapeutic agent is an antimitoticalkaloid. In general, antimitotic alkaloids can be extracted fromCantharanthus roseus, and have been shown to be efficacious asanticancer chemotherapy agents. A great number of semi-syntheticderivatives have been studied both chemically and pharmacologically(see, O. Van Tellingen et al, Anticancer Research, 12, 1699-1716(1992)). The antimitotic alkaloids of the present invention include, butare not limited to, vinblastine, vincristine, vindesine, Taxol andvinorelbine. The latter two antimitotic alkaloids are commerciallyavailable from Eli Lilly and Company, and Pierre Fabre Laboratories,respectively (see, U.S. Pat. No. 5,620,985). In a preferred aspect ofthe present invention, the antimitotic alkaloid is vinorelbine.

In other embodiments of the invention, the chemotherapeutic agent is adifluoronucleoside. 2′-deoxy-2′,2′-difluoronucleosides are known in theart as having antiviral activity. Such compounds are disclosed andtaught in U.S. Pat. Nos. 4,526,988 and 4,808,614. European PatentApplication Publication 184,365 discloses that these samedifluoronucleosides have oncolytic activity. In certain specificaspects, the 2′-deoxy-2′,2′-difluoronucleoside used in the compositionsand methods of the present invention is 2′-deoxy-2′,2′-difluorocytidinehydrochloride, also known as gemcitabine hydrochloride. Gemcitabine iscommercially available or can be synthesized in a multi-step process asdisclosed and taught in U.S. Pat. Nos. 4,526,988, 4,808,614 and5,223,608, the teachings of which are incorporated herein by reference.

Conjugates: Targeted Forms

One of ordinary skill in the art will readily appreciate that thecompounds of the present disclosure can be modified in any number ofways, such that the therapeutic or prophylactic efficacy of the compoundof the invention is increased through the modification. For instance,the compound of the present disclosure can be conjugated either directlyor indirectly through a linker to a targeting moiety. The practice ofconjugating compounds to targeting moieties is known in the art. See,for instance, Wadhwa et al., J Drug Targeting, 3, 111-127 (1995) andU.S. Pat. No. 5,087,616. The term “targeting moiety” as used herein,refers to any molecule or agent that specifically recognizes and bindsto a cell-surface receptor, such that the targeting moiety directs thedelivery of the compound of the invention to a population of cells onwhich surface the receptor is expressed. Targeting moieties include, butare not limited to, antibodies, or fragments thereof, peptides,hormones, growth factors, cytokines, and any other natural ornon-natural ligands, which bind to cell surface receptors (e.g.,Epithelial Growth Factor Receptor (EGFR), T-cell receptor (TCR), B-cellreceptor (BCR), CD28, Platelet-derived Growth Factor Receptor (PDGF),nicotinic acetylcholine receptor (nAChR), etc.). As used herein a“linker” is a bond, molecule or group of molecules that binds twoseparate entities to one another. Linkers may provide for optimalspacing of the two entities or may further supply a labile linkage thatallows the two entities to be separated from each other. Labile linkagesinclude photocleavable groups, acid-labile moieties, base-labilemoieties and enzyme-cleavable groups. The term “linker” in someembodiments refers to any agent or molecule that bridges the compound ofthe invention to the targeting moiety. The linker may be any of thosedescribed herein under the section entitled “Linkers.” One of ordinaryskill in the art recognizes that sites on the compound of the invention,which are not necessary for the function of the compound, are idealsites for attaching a linker and/or a targeting moiety, provided thatthe linker and/or targeting moiety, once attached to the compound,do(es) not interfere with the function of the compound, i.e., theability to inhibit the binding interaction between β integrin and Gprotein α subunit, as described herein.

Linkers

In some embodiments, the conjugate comprises a linker that joins thecompound of the invention to the heterologous moiety. In some aspects,the linker comprises a chain of atoms from 1 to about 60, or 1 to 30atoms or longer, 2 to 5 atoms, 2 to 10 atoms, 5 to 10 atoms, or 10 to 20atoms long. In some embodiments, the chain atoms are all carbon atoms.In some embodiments, the chain atoms in the backbone of the linker areselected from the group consisting of C, O, N, and S. Chain atoms andlinkers may be selected according to their expected solubility(hydrophilicity) so as to provide a more soluble conjugate. In someembodiments, the linker provides a functional group that is subject tocleavage by an enzyme or other catalyst or hydrolytic conditions foundin the target tissue or organ or cell. In some embodiments, the lengthof the linker is long enough to reduce the potential for sterichindrance. In some embodiments, the linker is an amino acid or apeptidyl linker. Such peptidyl linkers may be any length. Exemplarylinkers are from about 1 to 50 amino acids in length, 5 to 50, 3 to 5, 5to 10, 5 to 15, or 10 to 30 amino acids in length.

Dimers & Multimers

In some embodiments, the compound is provided as a dimer or a multimerin which more than one compound of the invention are linked together.The dimer in some aspects is a homodimer comprising two compounds of thesame type (e.g., same structure) linked together. In alternativeaspects, the dimer is a heterodimer comprising two compounds of theinvention, wherein the two compounds are structurally distinct from eachother. The multimer in some aspects is a homomultimer comprising morethan one compound of the invention and each compound are of the sametype (e.g., same structure). In alternative aspects, the multimer is aheteromultimer comprising more than one compound of the invention andwherein at least two compounds of the heteromultimer are structurallydistinct from the other. Two or more of the compounds can be linkedtogether using standard linking agents and procedures known to thoseskilled in the art. In certain embodiments, the linker connecting thetwo (or more) compounds is a linker as described in the section entitled“Linkers.” In some embodiments, the linker is a disulfide bond. Forexample, each monomer of the dimer may comprise a sulfhydryl and thesulfur atom of each participates in the formation of the disulfide bond.

Compositions

The invention further provide compositions comprising a compound thatinhibits a binding interaction between a β integrin and a G protein αsubunit, e.g., an antibody, antigen binding fragment, aptamer, peptide,peptide analog, pharmaceutically acceptable salt, conjugate, multimer,dimer, as described herein. The compositions in some aspects comprisethe compounds in isolated and/or purified form. In some aspects, thecomposition comprises a single type (e.g., structure) of a compound ofthe invention or comprises a combination of two or more compounds of theinvention, wherein the combination comprises two or more compounds ofdifferent types (e.g., structures).

In some aspects, the composition comprises agents which enhance thechemico-physico features of the compound, e.g., via stabilizing thecompound at certain temperatures, e.g., room temperature, increasingshelf life, reducing degradation, e.g., oxidation protease mediateddegradation, increasing half-life of the compound, etc. In some aspects,the composition comprises any of the agents disclosed herein as aheterologous moiety or conjugate moiety, optionally in admixture withthe compounds of the invention or conjugated to the compounds.

In certain aspects, the composition comprises a delivery agent whichaids in localizing the compound of the invention to the appropriateplace. In exemplary embodiments, the composition comprises a vehiclewhich aids in getting the compound of the invention inside a cell. Inexemplary aspects, the vehicle is covalently attached to the compound.In alternative aspects, the composition comprises a vehicle in admixturewith the compound. In exemplary aspects, the vehicle comprises or is anyof the heterologous moieties described herein with regard to conjugates.For example, the vehicle may be a polymer, e.g., water soluble polymer,which may be linear or branched. In exemplary aspects, the the watersoluble polymer is selected from the group consisting of polyethyleneglycol (PEG), branched PEG, polysialic acid (PSA), carbohydrate,polysaccharides, pullulane, chitosan, hyaluronic acid, chondroitinsulfate, dermatan sulfate, starch or a starch derivative, dextran,carboxymethyl-dextran, polyalkylene oxide (PAO), polyalkylene glycol(PAG), polypropylene glycol (PPG), polyoxazoline, polyacryloylmorpholine, polyvinyl alcohol (PVA), polycarboxylate,polyvinylpyrrolidone, polyphosphazene, polyoxazoline,polyethylene-co-maleic acid anhydride, polystyrene-co-maleic acidanhydride, poly(1-hydroxymethylethylene hydroxymethylformal) (PHF), and2-methacryloyloxy-2′-ethyltrimethylammoniumphosphate (MPC).

In exemplary embodiments, the vehicle comprises a carbohydrate, such asany of those described herein. In exemplary aspects, the vehiclecomprises a polysaccharide.

In exemplary aspects, the vehicle comprises a lipophilic moiety. Inexemplary aspects, the vehicle comprises a fatty acid. The fatty acidmay be a C4 to C30 fatty acid, e.g., C4 fatty acid, C6 fatty acid, C8fatty acid, C10 fatty acid, C12 fatty acid, C14 fatty acid, C16 fattyacid, C18 fatty acid, C20 fatty acid, C22 fatty acid, C24 fatty acid,C26 fatty acid, C28 fatty acid, or a C30 fatty acid. In exemplaryaspects, the fatty acid is a C12 to C30 fatty acid. In exemplaryaspects, the fatty acid is a myristoyl group or a palmitoyl group. Inexemplary aspects, the compound is a peptide or peptide analog and thefatty acid is covalently attached to the peptide or peptide analog. Forexample, the fatty acid is covalently attached to the peptide or peptideanalog at the N-terminus or C-terminus or via a side chain of anon-terminal amino acid of the peptide or peptide analog.

In exemplary aspects, the vehicle comprises a polypeptide, which when inthe composition improves the ability of the composition to enter a cellcompared to the ability of the composition in the absence of thepolypeptide. In certain aspects, the composition comprises a compound ofthe invention (e.g., a peptide that inhibits a binding interactionbetween a β integrin and a G protein α subunit) and a peptide deliveryagent. In some aspects, the peptide delivery agent is a cell penetratingpeptide (CPP). In particular aspects, the composition comprises a CPPfused to the compound, e.g., the composition comprises a fusion peptideproduct comprising a peptide of the invention that inhibits a bindinginteraction between a β integrin and a G protein a subunit fused to aCPP.

Pharmaceutical Compositions and Formulations

In yet other aspects of the invention, the composition comprises acompound that inhibits a binding interaction between a β integrin and aG protein α subunit and additionally comprises a pharmaceuticallyacceptable carrier, diluents, or excipient. In some embodiments, thecompound of the invention, the pharmaceutically acceptable salt, theconjugate, the dimer or multimer, of the invention (hereinafter referredto as “active agents”) is formulated into a pharmaceutical compositioncomprising the the active agent, along with a pharmaceuticallyacceptable carrier, diluent, or excipient. In this regard, the inventionfurther provides pharmaceutical compositions comprising an active agentthat inhibits a binding interaction between a β integrin and a G proteinα subunit which is intended for administration to a subject, e.g., amammal.

In some embodiments, the active agent is present in the pharmaceuticalcomposition at a purity level suitable for administration to a patient.In some embodiments, the active agent has a purity level of at leastabout 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about96%, about 97%, about 98% or about 99%, and a pharmaceuticallyacceptable diluent, carrier or excipient. The pharmaceutical compositionin some aspects comprises the active agent of the present disclosure ata concentration of at least A, wherein A is about 0.001 mg/ml, about0.01 mg/ml, 0 about 1 mg/ml, about 0.5 mg/ml, about 1 mg/ml, about 2mg/ml, about 3 mg/ml, about 4 mg/ml, about 5 mg/ml, about 6 mg/ml, about7 mg/ml, about 8 mg/ml, about 9 mg/ml, about 10 mg/ml, about 11 mg/ml,about 12 mg/ml, about 13 mg/ml, about 14 mg/ml, about 15 mg/ml, about 16mg/ml, about 17 mg/ml, about 18 mg/ml, about 19 mg/ml, about 20 mg/ml,about 21 mg/ml, about 22 mg/ml, about 23 mg/ml, about 24 mg/ml, about 25mg/ml or higher. In some embodiments, the pharmaceutical compositioncomprises the active agent at a concentration of at most B, wherein B isabout 30 mg/ml, about 25 mg/ml, about 24 mg/ml, about 23, mg/ml, about22 mg/ml, about 21 mg/ml, about 20 mg/ml, about 19 mg/ml, about 18mg/ml, about 17 mg/ml, about 16 mg/ml, about 15 mg/ml, about 14 mg/ml,about 13 mg/ml, about 12 mg/ml, about 11 mg/ml, about 10 mg/ml, about 9mg/ml, about 8 mg/ml, about 7 mg/ml, about 6 mg/ml, about 5 mg/ml, about4 mg/ml, about 3 mg/ml, about 2 mg/ml, about 1 mg/ml, or about 0.1mg/ml. In some embodiments, the compositions may contain an active agentat a concentration range of A to B mg/ml, for example, about 0.001 toabout 30.0 mg/ml.

Depending on the route of administration, the particular active agentintended for use, as well as other factors, the pharmaceuticalcomposition may comprise additional pharmaceutically acceptableingredients, including, for example, acidifying agents, additives,adsorbents, aerosol propellants, air displacement agents, alkalizingagents, anticaking agents, anticoagulants, antimicrobial preservatives,antioxidants, antiseptics, bases, binders, buffering agents, chelatingagents, coating agents, coloring agents, desiccants, detergents,diluents, disinfectants, disintegrants, dispersing agents, dissolutionenhancing agents, dyes, emollients, emulsifying agents, emulsionstabilizers, fillers, film forming agents, flavor enhancers, flavoringagents, flow enhancers, gelling agents, granulating agents, humectants,lubricants, mucoadhesives, ointment bases, ointments, oleaginousvehicles, organic bases, pastille bases, pigments, plasticizers,polishing agents, preservatives, sequestering agents, skin penetrants,solubilizing agents, solvents, stabilizing agents, suppository bases,surface active agents, surfactants, suspending agents, sweeteningagents, therapeutic agents, thickening agents, tonicity agents, toxicityagents, viscosity-increasing agents, water-absorbing agents,water-miscible cosolvents, water softeners, or wetting agents.

Accordingly, in some embodiments, the pharmaceutical compositioncomprises any one or a combination of the following components: acacia,acesulfame potassium, acetyltributyl citrate, acetyltriethyl citrate,agar, albumin, alcohol, dehydrated alcohol, denatured alcohol, dilutealcohol, aleuritic acid, alginic acid, aliphatic polyesters, alumina,aluminum hydroxide, aluminum stearate, amylopectin, α-amylose, ascorbicacid, ascorbyl palmitate, aspartame, bacteriostatic water for injection,bentonite, bentonite magma, benzalkonium chloride, benzethoniumchloride, benzoic acid, benzyl alcohol, benzyl benzoate, bronopol,butylated hydroxyanisole, butylated hydroxytoluene, butylparaben,butylparaben sodium, calcium alginate, calcium ascorbate, calciumcarbonate, calcium cyclamate, dibasic anhydrous calcium phosphate,dibasic dehydrate calcium phosphate, tribasic calcium phosphate, calciumpropionate, calcium silicate, calcium sorbate, calcium stearate, calciumsulfate, calcium sulfate hemihydrate, canola oil, carbomer, carbondioxide, carboxymethyl cellulose calcium, carboxymethyl cellulosesodium, β-carotene, carrageenan, castor oil, hydrogenated castor oil,cationic emulsifying wax, cellulose acetate, cellulose acetatephthalate, ethyl cellulose, microcrystalline cellulose, powderedcellulose, silicified microcrystalline cellulose, sodium carboxymethylcellulose, cetostearyl alcohol, cetrimide, cetyl alcohol, chlorhexidine,chlorobutanol, chlorocresol, cholesterol, chlorhexidine acetate,chlorhexidine gluconate, chlorhexidine hydrochloride,chlorodifluoroethane (HCFC), chlorodifluoromethane, chlorofluorocarbons(CFC)chlorophenoxyethanol, chloroxylenol, corn syrup solids, anhydrouscitric acid, citric acid monohydrate, cocoa butter, coloring agents,corn oil, cottonseed oil, cresol, m-cresol, o-cresol, p-cresol,croscarmellose sodium, crospovidone, cyclamic acid, cyclodextrins,dextrates, dextrin, dextrose, dextrose anhydrous, diazolidinyl urea,dibutyl phthalate, dibutyl sebacate, diethanolamine, diethyl phthalate,difluoroethane (HFC), dimethyl-β-cyclodextrin, cyclodextrin-typecompounds such as Captisol®, dimethyl ether, dimethyl phthalate,dipotassium edentate, disodium edentate, disodium hydrogen phosphate,docusate calcium, docusate potassium, docusate sodium, dodecyl gallate,dodecyltrimethylammonium bromide, edentate calcium disodium, edtic acid,eglumine, ethyl alcohol, ethylcellulose, ethyl gallate, ethyl laurate,ethyl maltol, ethyl oleate, ethylparaben, ethylparaben potassium,ethylparaben sodium, ethyl vanillin, fructose, fructose liquid, fructosemilled, fructose pyrogen-free, powdered fructose, fumaric acid, gelatin,glucose, liquid glucose, glyceride mixtures of saturated vegetable fattyacids, glycerin, glyceryl behenate, glyceryl monooleate, glycerylmonostearate, self-emulsifying glyceryl monostearate, glycerylpalmitostearate, glycine, glycols, glycofurol, guar gum,heptafluoropropane (HFC), hexadecyltrimethylammonium bromide, highfructose syrup, human serum albumin, hydrocarbons (HC), dilutehydrochloric acid, hydrogenated vegetable oil, type II, hydroxyethylcellulose, 2-hydroxyethyl-β-cyclodextrin, hydroxypropyl cellulose,low-substituted hydroxypropyl cellulose, 2-hydroxypropyl-β-cyclodextrin,hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate,imidurea, indigo carmine, ion exchangers, iron oxides, isopropylalcohol, isopropyl myristate, isopropyl palmitate, isotonic saline,kaolin, lactic acid, lactitol, lactose, lanolin, lanolin alcohols,anhydrous lanolin, lecithin, magnesium aluminum silicate, magnesiumcarbonate, normal magnesium carbonate, magnesium carbonate anhydrous,magnesium carbonate hydroxide, magnesium hydroxide, magnesium laurylsulfate, magnesium oxide, magnesium silicate, magnesium stearate,magnesium trisilicate, magnesium trisilicate anhydrous, malic acid,malt, maltitol, maltitol solution, maltodextrin, maltol, maltose,mannitol, medium chain triglycerides, meglumine, menthol,methylcellulose, methyl methacrylate, methyl oleate, methylparaben,methylparaben potassium, methylparaben sodium, microcrystallinecellulose and carboxymethylcellulose sodium, mineral oil, light mineraloil, mineral oil and lanolin alcohols, oil, olive oil, monoethanolamine,montmorillonite, octyl gallate, oleic acid, palmitic acid, paraffin,peanut oil, petrolatum, petrolatum and lanolin alcohols, pharmaceuticalglaze, phenol, liquified phenol, phenoxyethanol, phenoxypropanol,phenylethyl alcohol, phenylmercuric acetate, phenylmercuric borate,phenylmercuric nitrate, polacrilin, polacrilin potassium, poloxamer,polydextrose, polyethylene glycol, polyethylene oxide, polyacrylates,polyethylene-polyoxypropylene-block polymers, polymethacrylates,polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives,polyoxyethylene sorbitol fatty acid esters, polyoxyethylene stearates,polyvinyl alcohol, polyvinyl pyrrolidone, potassium alginate, potassiumbenzoate, potassium bicarbonate, potassium bisulfite, potassiumchloride, postassium citrate, potassium citrate anhydrous, potassiumhydrogen phosphate, potassium metabisulfite, monobasic potassiumphosphate, potassium propionate, potassium sorbate, povidone, propanol,propionic acid, propylene carbonate, propylene glycol, propylene glycolalginate, propyl gallate, propylparaben, propylparaben potassium,propylparaben sodium, protamine sulfate, rapeseed oil, Ringer'ssolution, saccharin, saccharin ammonium, saccharin calcium, saccharinsodium, safflower oil, saponite, serum proteins, sesame oil, colloidalsilica, colloidal silicon dioxide, sodium alginate, sodium ascorbate,sodium benzoate, sodium bicarbonate, sodium bisulfite, sodium chloride,anhydrous sodium citrate, sodium citrate dehydrate, sodium chloride,sodium cyclamate, sodium edentate, sodium dodecyl sulfate, sodium laurylsulfate, sodium metabisulfite, sodium phosphate, dibasic, sodiumphosphate, monobasic, sodium phosphate, tribasic, anhydrous sodiumpropionate, sodium propionate, sodium sorbate, sodium starch glycolate,sodium stearyl fumarate, sodium sulfite, sorbic acid, sorbitan esters(sorbitan fatty esters), sorbitol, sorbitol solution 70%, soybean oil,spermaceti wax, starch, corn starch, potato starch, pregelatinizedstarch, sterilizable maize starch, stearic acid, purified stearic acid,stearyl alcohol, sucrose, sugars, compressible sugar, confectioner'ssugar, sugar spheres, invert sugar, Sugartab, Sunset Yellow FCF,synthetic paraffin, talc, tartaric acid, tartrazine, tetrafluoroethane(HFC), theobroma oil, thimerosal, titanium dioxide, alpha tocopherol,tocopheryl acetate, alpha tocopheryl acid succinate, beta-tocopherol,delta-tocopherol, gamma-tocopherol, tragacanth, triacetin, tributylcitrate, triethanolamine, triethyl citrate, trimethyl-β-cyclodextrin,trimethyltetradecylammonium bromide, tris buffer, trisodium edentate,vanillin, type I hydrogenated vegetable oil, water, soft water, hardwater, carbon dioxide-free water, pyrogen-free water, water forinjection, sterile water for inhalation, sterile water for injection,sterile water for irrigation, waxes, anionic emulsifying wax, carnaubawax, cationic emulsifying wax, cetyl ester wax, microcrystalline wax,nonionic emulsifying wax, suppository wax, white wax, yellow wax, whitepetrolatum, wool fat, xanthan gum, xylitol, zein, zinc propionate, zincsalts, zinc stearate, or any excipient in the Handbook of PharmaceuticalExcipients, Third Edition, A. H. Kibbe (Pharmaceutical Press, London,UK, 2000), which is incorporated by reference in its entirety.Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin(Mack Publishing Co., Easton, Pa., 1980), which is incorporated byreference in its entirety, discloses various components used informulating pharmaceutically acceptable compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional agent is incompatible with the pharmaceutical compositions,its use in pharmaceutical compositions is contemplated. Supplementaryactive ingredients also can be incorporated into the compositions.

In some embodiments, the foregoing component(s) may be present in thepharmaceutical composition at any concentration, such as, for example,at least A, wherein A is 0.0001% w/v, 0.001% w/v, 0.01% w/v, 0.1% w/v,1% w/v, 2% w/v, 5% w/v, 10% w/v, 20% w/v, 30% w/v, 40% w/v, 50% w/v, 60%w/v, 70% w/v, 80% w/v, or 90% w/v. In some embodiments, the foregoingcomponent(s) may be present in the pharmaceutical composition at anyconcentration, such as, for example, at most B, wherein B is 90% w/v,80% w/v, 70% w/v, 60% w/v, 50% w/v, 40% w/v, 30% w/v, 20% w/v, 10% w/v,5% w/v, 2% w/v, 1% w/v, 0.1% w/v, 0.001% w/v, or 0.0001%. In otherembodiments, the foregoing component(s) may be present in thepharmaceutical composition at any concentration range, such as, forexample from about A to about B. In some embodiments, A is 0.0001% and Bis 90%.

The pharmaceutical compositions may be formulated to achieve aphysiologically compatible pH. In some embodiments, the pH of thepharmaceutical composition may be at least 5, at least 5.5, at least 6,at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, atleast 9, at least 9.5, at least 10, or at least 10.5 up to and includingpH 11, depending on the formulation and route of administration. Incertain embodiments, the pharmaceutical compositions may comprisebuffering agents to achieve a physiological compatible pH. The bufferingagents may include any compounds capabale of buffering at the desired pHsuch as, for example, phosphate buffers (e.g., PBS), triethanolamine,Tris, bicine, TAPS, tricine, HEPES, TES, MOPS, PIPES, cacodylate, MES,and others. In certain embodiments, the strength of the buffer is atleast 0.5 mM, at least 1 mM, at least 5 mM, at least 10 mM, at least 20mM, at least 30 mM, at least 40 mM, at least 50 mM, at least 60 mM, atleast 70 mM, at least 80 mM, at least 90 mM, at least 100 mM, at least120 mM, at least 150 mM, or at least 200 mM. In some embodiments, thestrength of the buffer is no more than 300 mM (e.g., at most 200 mM, atmost 100 mM, at most 90 mM, at most 80 mM, at most 70 mM, at most 60 mM,at most 50 mM, at most 40 mM, at most 30 mM, at most 20 mM, at most 10mM, at most 5 mM, at most 1 mM).

Routes of Administration

With regard to the invention, the active agent, pharmaceuticalcomposition comprising the same, may be administered to the subject viaany suitable route of administration. The following discussion on routesof administration is merely provided to illustrate exemplary embodimentsand should not be construed as limiting the scope in any way.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the active agent of thepresent disclosure dissolved in diluents, such as water, saline, ororange juice; (b) capsules, sachets, tablets, lozenges, and troches,each containing a predetermined amount of the active ingredient, assolids or granules; (c) powders; (d) suspensions in an appropriateliquid; and (e) suitable emulsions. Liquid formulations may includediluents, such as water and alcohols, for example, ethanol, benzylalcohol, and the polyethylene alcohols, either with or without theaddition of a pharmaceutically acceptable surfactant. Capsule forms canbe of the ordinary hard- or soft-shelled gelatin type containing, forexample, surfactants, lubricants, and inert fillers, such as lactose,sucrose, calcium phosphate, and corn starch. Tablet forms can includeone or more of lactose, sucrose, mannitol, corn starch, potato starch,alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum,colloidal silicon dioxide, croscarmellose sodium, talc, magnesiumstearate, calcium stearate, zinc stearate, stearic acid, and otherexcipients, colorants, diluents, buffering agents, disintegratingagents, moistening agents, preservatives, flavoring agents, and otherpharmacologically compatible excipients. Lozenge forms can comprise theactive agent of the present disclosure in a flavor, usually sucrose andacacia or tragacanth, as well as pastilles comprising the active agentof the present disclosure in an inert base, such as gelatin andglycerin, or sucrose and acacia, emulsions, gels, and the likecontaining, in addition to, such excipients as are known in the art.

The active agents of the present disclosure, alone or in combinationwith other suitable components, can be delivered via pulmonaryadministration and can be made into aerosol formulations to beadministered via inhalation. These aerosol formulations can be placedinto pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like. They also maybe formulated as pharmaceuticals for non-pressured preparations, such asin a nebulizer or an atomizer. Such spray formulations also may be usedto spray mucosa. In some embodiments, the active agent is formulatedinto a powder blend or into microparticles or nanoparticles. Suitablepulmonary formulations are known in the art. See, e.g., Qian et al., IntJ Pharm 366: 218-220 (2009); Adjei and Garren, Pharmaceutical Research,7(6): 565-569 (1990); Kawashima et al., J Controlled Release 62(1-2):279-287 (1999); Liu et al., Pharm Res 10(2): 228-232 (1993);International Patent Application Publication Nos. WO 2007/133747 and WO2007/141411.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The term, “parenteral” means not through the alimentary canal but bysome other route such as subcutaneous, intramuscular, intraspinal, orintravenous. The active agent of the present disclosure can beadministered with a physiologically acceptable diluent in apharmaceutical carrier, such as a sterile liquid or mixture of liquids,including water, saline, aqueous dextrose and related sugar solutions,an alcohol, such as ethanol or hexadecyl alcohol, a glycol, such aspropylene glycol or polyethylene glycol, dimethylsulfoxide, glycerol,ketals such as 2,2-dimethyl-153-dioxolane-4-methanol, ethers,poly(ethyleneglycol) 400, oils, fatty acids, fatty acid esters orglycerides, or acetylated fatty acid glycerides with or without theaddition of a pharmaceutically acceptable surfactant, such as a soap ora detergent, suspending agent, such as pectin, carbomers,methylcellulose, hydroxypropylmethylcellulose, orcarboxymethylcellulose, or emulsifying agents and other pharmaceuticaladjuvants.

Oils, which can be used in parenteral formulations include petroleum,animal, vegetable, or synthetic oils. Specific examples of oils includepeanut, soybean, sesame, cottonseed, corn, olive, petrolatum, andmineral. Suitable fatty acids for use in parenteral formulations includeoleic acid, stearic acid, and isostearic acid. Ethyl oleate andisopropyl myristate are examples of suitable fatty acid esters.

Suitable soaps for use in parenteral formulations include fatty alkalimetal, ammonium, and triethanolamine salts, and suitable detergentsinclude (a) cationic detergents such as, for example, dimethyl dialkylammonium halides, and alkyl pyridinium halides, (b) anionic detergentssuch as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin,ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionicdetergents such as, for example, fatty amine oxides, fatty acidalkanolamides, and polyoxyethylenepolypropylene copolymers, (d)amphoteric detergents such as, for example, alkyl-β-aminopropionates,and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixturesthereof.

The parenteral formulations in some embodiments contain from about 0.5%to about 25% by weight of the active agent of the present disclosure insolution. Preservatives and buffers may be used. In order to minimize oreliminate irritation at the site of injection, such compositions maycontain one or more nonionic surfactants having a hydrophile-lipophilebalance (HLB) of from about 12 to about 17. The quantity of surfactantin such formulations will typically range from about 5% to about 15% byweight. Suitable surfactants include polyethylene glycol sorbitan fattyacid esters, such as sorbitan monooleate and the high molecular weightadducts of ethylene oxide with a hydrophobic base, formed by thecondensation of propylene oxide with propylene glycol. The parenteralformulations in some aspects are presented in unit-dose or multi-dosesealed containers, such as ampoules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions in some aspects are prepared from sterile powders, granules,and tablets of the kind previously described.

Injectable formulations are in accordance with the invention. Therequirements for effective pharmaceutical carriers for injectablecompositions are well-known to those of ordinary skill in the art (see,e.g., Pharmaceutics and Pharmacy Practice, J. B. Lippincott Company,Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), andASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630(1986)).

Additionally, the active agent of the invention can be made intosuppositories for rectal administration by mixing with a variety ofbases, such as emulsifying bases or water-soluble bases. Formulationssuitable for vaginal administration can be presented as pessaries,tampons, creams, gels, pastes, foams, or spray formulas containing, inaddition to the active ingredient, such carriers as are known in the artto be appropriate.

It will be appreciated by one of skill in the art that, in addition tothe above-described pharmaceutical compositions, the active agent of thedisclosure can be formulated as inclusion complexes, such ascyclodextrin inclusion complexes, or liposomes.

Dosages

The active agents of the disclosure are believed to be useful in methodsof inhibiting a binding interaction between β integrin and G protein αsubunit, as well as other methods, as further described herein,including methods of treating or preventing stroke, heart attack,cancer, or inflammation. For purposes of the disclosure, the amount ordose of the active agent administered should be sufficient to effect,e.g., a therapeutic or prophylactic response, in the subject or animalover a reasonable time frame. For example, the dose of the active agentof the present disclosure should be sufficient to treat cancer asdescribed herein in a period of from about 1 to 4 minutes, 1 to 4 hoursor 1 to 4 weeks or longer, e.g., 5 to 20 or more weeks, from the time ofadministration. In certain embodiments, the time period could be evenlonger. The dose will be determined by the efficacy of the particularactive agent and the condition of the animal (e.g., human), as well asthe body weight of the animal (e.g., human) to be treated.

Many assays for determining an administered dose are known in the art.For purposes herein, an assay, which comprises comparing the extent towhich cancer is treated upon administration of a given dose of theactive agent of the present disclosure to a mammal among a set ofmammals, each set of which is given a different dose of the activeagent, could be used to determine a starting dose to be administered toa mammal. The extent to which cancer is treated upon administration of acertain dose can be represented by, for example, the cytotoxicity of theactive agent or the extent of tumor regression achieved with the activeagent in a mouse xenograft model. Methods of measuring cytotoxicity ofcompounds and methods of assaying tumor regression are known in the art,including, for instance, the methods described in the EXAMPLES set forthbelow.

The dose of the active agent of the present disclosure also will bedetermined by the existence, nature and extent of any adverse sideeffects that might accompany the administration of a particular activeagent of the present disclosure. Typically, the attending physician willdecide the dosage of the active agent of the present disclosure withwhich to treat each individual patient, taking into consideration avariety of factors, such as age, body weight, general health, diet, sex,active agent of the present disclosure to be administered, route ofadministration, and the severity of the condition being treated. By wayof example and not intending to limit the invention, the dose of theactive agent of the present disclosure can be about 0.0001 to about 1g/kg body weight of the subject being treated/day, from about 0.0001 toabout 0.001 g/kg body weight/day, or about 0.01 mg to about 1 g/kg bodyweight/day.

Controlled Release Formulations

In some embodiments, the active agents described herein can be modifiedinto a depot form, such that the manner in which the active agent of theinvention is released into the body to which it is administered iscontrolled with respect to time and location within the body (see, forexample, U.S. Pat. No. 4,450,150). Depot forms of active agents of theinvention can be, for example, an implantable composition comprising theactive agents and a porous or non-porous material, such as a polymer,wherein the active agent is encapsulated by or diffused throughout thematerial and/or degradation of the non-porous material. The depot isthen implanted into the desired location within the body of the subjectand the active agent is released from the implant at a predeterminedrate.

The pharmaceutical composition comprising the active agent in certainaspects is modified to have any type of in vivo release profile. In someaspects, the pharmaceutical composition is an immediate release,controlled release, sustained release, extended release, delayedrelease, or bi-phasic release formulation. Methods of formulatingpeptides for controlled release are known in the art. See, for example,Qian et al., J Pharm 374: 46-52 (2009) and International PatentApplication Publication Nos. WO 2008/130158, WO2004/033036;WO2000/032218; and WO 1999/040942.

The instant compositions may further comprise, for example, micelles orliposomes, or some other encapsulated form, or may be administered in anextended release form to provide a prolonged storage and/or deliveryeffect.

Micelles

The invention also provides a micelle comprising a peptide or peptideanalog of the invention and at least one lipid, optionally, wherein thelipid is covalently attached to a water soluble polymer. In exemplaryaspects, the peptide or peptide analog of the micelle is covalentlyattached to a fatty acid or other lipid moiety. In exemplary aspects,the peptide of the micelle consists of FEEERA (SEQ ID NO: 87), whereinthe Phe at position 1 is covalently attached to a C16 fatty acid. Inexemplary aspects, the micelle comprises a lipid covalently attached toa water soluble polymer and a lipid free of a water soluble polymer.Suitable lipids for use in micelle synthesis is known in the art. See,e.g., Banerjee and Onyuksel, Peptide Delivery Using PhospholipidMicelles, WIREs Nanomed Nanobiotechnol 4:562-574 (2012). In exemplaryaspects, the lipid that is covalently attached to a water solublepolymer is1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000] and the lipid that is free of a water soluble polymer isphophatidylcholine.

The invention further comprises a composition comprising any of themicelles described herein and an aqueous solution.

Timing of Administration

The disclosed pharmaceutical compositions and formulations may beadministered according to any regimen including, for example, daily (1time per day, 2 times per day, 3 times per day, 4 times per day, 5 timesper day, 6 times per day), every two days, every three days, every fourdays, every five days, every six days, weekly, bi-weekly, every threeweeks, monthly, or bi-monthly. Timing, like dosing can be fine-tunedbased on dose-response studies, efficacy, and toxicity data, andinitially guaged based on timing used for other antibody therapeutics.

Combinations

In some embodiments, the active agents described herein are administeredalone, and in alternative embodiments, the active agents describedherein are administered in combination with another therapeutic agent,e.g., another active agent of the invention of different type (e.g.,structure), or another therapeutic which does not inhibit a bindinginteraction between β integrin and G protein α subunit. In some aspects,the other therapeutic aims to treat or prevent cancer. In specificaspects, the other therapeutic is one listed under the section entitled“Heterologous Moieties: Therapeutic Agents.” In some embodiments, theother therapeutic is a chemotherapeutic agent. In some embodiments, theother therapeutic is an agent used in radiation therapy for thetreatment of cancer.

In exemplary aspects, the active agents described herein areadministered or packaged in combination with an anti-thrombotic agent.In exemplary embodiments, the anti-thrombotic agent is an anticoagulant,e.g., fondaparinux and bivalirudin. In exemplary embodiments, theanti-thrombotic agent is an anti-platelet agent, e.g., aspirin,clopidogrel, dipyridamole, and abciximab.

In exemplary aspects, the active agent described herein is administeredor packaged in combination with an anti-platelet drug. In exemplaryaspects, the antiplatelet drug is an irreversible cyclooxygenaseinhibitor (e.g., aspirin), an adenosine diphosphate (ADP) receptorinhibitor (e.g., clopidogrel, prasugrel, ticagrelor, ticlopidine), aphosphodiesterase inhibitor (e.g., cilostazol), a glycoprotein IIb/IIIainhibitor (e.g., abciximab, eptifibatide), tirofiban), an adenosinereuptake inhibitor (e.g., dipyridamole), or a thromboxane inhibitor(e.g., a thromboxane synthase inhibitor, a thromboxane receptorantagonist (e.g., terutroban). In exemplary aspects, the anti-plateletdrug is aspirin, a thienopyridine, a cylooxygenase inhibitor or a P2Y12inhibitor.

In exemplary aspects, the active agent described herein is administeredor packaged in combination with an integrin antagonist or integrininhibitor. The integrin inhibitor in exemplary aspects is eptifibatide

In exemplary embodiments, the active agent is administeredsimultaneously as the other therapeutic. In alternative embodiments, theactive agent is administered either before or after the othertherapeutic.

Methods of Use

Given the importance of the biological roles of a β integrin and a Gprotein α subunit, individually, and, as shown herein, in combinationwith one another, the active agents of the invention are useful for anumber of applications in a variety of settings. For example and mostsimplistically, the active agents of the invention are useful forinhibiting a binding interaction between a β integrin and a G protein αsubunit in a cell. In this regard, the invention provide a method ofinhibiting a binding interaction between a β integrin and a G protein αsubunit in a cell. The method comprises contacting the cell with acompound or composition of the invention, in an amount effective toinhibit the binding interaction. In some aspects, the cell is part of anin vitro or ex vivo cell culture or in vitro or ex vivo tissue sample.In some aspects, the cell is an in vivo cell. In certain embodiments,the method is intended for research purposes, and, in other embodiments,the method is intended for therapeutic purposes.

The invention also provides a method of inhibiting integrin-dependentSrc activation in a cell. The method comprises the step of contactingthe cells with a compound or composition of the invention in an amounteffective to inhibiting the Src activation. Methods of measuringintegrin-dependent Src activation are known in the art, and include, forexample, those set forth herein in EXAMPLES and use of the ProFluor®Src-Family Kinase Assay (Promega, Madison, Wis.) or one of the Srcactivity assay kits available from Millipore (Billerica, Mass.).

A method of activating a small GTPase is furthermore provided by theinvention. The method comprises the step of contacting a G proteinsubunit with a compound or composition in an amount effective toactivate the small GTPase. In exemplary aspects, the small GTPase of themethod of the invention is RhoA. Methods of measuring GTPase activityare known in the art, and include, for example, those set forth hereinin EXAMPLES. Additionally, the GTPase activity levels may be measuredusing commercially available kits (Thermo Fisher Scientific, Inc.(Rockford, Ill.), Innova Biosciences (Cambridge, UK), Cell Biolabs, Inc.(San Diego, Calif.).

The invention moreover provides a method of inhibiting spreading ormigration of a cell. The method comprises the step of contacting thecell with a compound or composition of the invention in an amounteffective to inhibit spreading and migration. The cell may be any cellthat undergoes integrin-dependent adhesion, spreading, retraction, ormigration, or any cell that undergoes anchorage-dependent survival andproliferation. In exemplary aspects, the cell is a platelet, leukocyte,endothelial cell, fibroblast, epithelial cell. Methods of measuring cellspreading or cell migration are known in the art. See, for example, themethods described herein in EXAMPLES.

Also provided by the invention is a method of inhibiting plateletadhesion. The method comprises the step of contacting a platelet with acompound or composition of the invention in an amount effective toinhibit platelet adhesion. The invention further provides a method ofinhibiting platelet granule secretion and platelet aggregation. Themethod comprises the step of contacting a platelet with a compound orcomposition of the invention in an amount effective to inhibit granulesecretion and aggregation. Methods of measuring platelet granulesecretion or platelet aggregation are known in the art. For example,platelet aggregation may be measured in platelet rich-plasma (PRP) usinga turbidomatric platelet aggregometer to analyze washed platelets.Aggregation may be indicated by an increase in light transmissionthrough PRP or platelet suspension. Platelet aggregation also may bemeasured in whole blood using a whole blood platelet aggregometer, inwhich platelet aggregation is indicated by an increase in electricresistance of electrodes. See, for example, the methods described hereinin EXAMPLES.

The compounds and compositions of the invention are additionallycontemplated for therapeutic purposes. For example, the compounds andcompositions of the invention may be used to enhance blood clotretraction or inhibit thrombosis in a subject in need thereof.Accordingly, the invention provides a method of enhancing blood clotretraction in a subject in need thereof. The method comprises the stepof administering to the subject a compound or composition of theinvention in an amount effective to enhance blood clot retraction. Alsoprovided is a method of inhibiting thrombosis in a subject in needthereof. The method comprises the step of administering to the subject acompound or composition of the invention in an amount effective toinhibit thrombosis.

Because blood clotting and thrombosis play a role in stroke and heartattack, the invention furthermore provides a method of treating orpreventing a stroke or a heart attack in a subject in need thereof. Themethod comprises the step of administering to the subject a compound orcomposition of the invention in an amount effective to treat or preventstroke or heart attack. Because the compounds and compositions providedherein relate to the coordinated cell spreading-retraction process,which in turn, is important in cell migration, the invention alsoprovides a method of inhibiting metastasis of a tumor cell. The methodcomprises the step of contacting a tumor cell with a compound orcomposition of the invention in an amount effective to inhibitmetastasis. The compounds and compositions are also contemplated for usein inhibiting angiogenesis. Accordingly, the invention provides a methodof inhibiting angiogenesis in a subject in need thereof. The methodcomprises the step of administering to the subject a compound orcomposition of the invention in an amount effective to inhibitangiogenesis.

Promotion of clot and cell retraction by the compounds of the inventioncan also facilitate wound healing. Accordingly, the invention provides amethod of facilitating wound healing in a subject in need of thereof.The method comprises the step of administrating to the subject of acompound or composition of of the invention in an amount effective tofacilitate the wound healing. In exemplary aspects, the compound orcomposition is administered to the subject topically near or at thewound site. In exemplary embodiments, the rate of wound healing isincreased by at least 10%, 25%, 50%, 75%, 90% or more, as compared tothe time at which the wound would heal without administration of thecompound or composition of the invention.

Since metastasis and angiogenesis are important aspects of cancer, theinvention moreover provides a method of treating or preventing cancer ina subject in need thereof. The method comprises the step ofadministering to the subject a compound or composition of the inventionin an amount effective to treat or prevent cancer. In exemplary aspects,the subject has a solid tumor and the compound or composition of theinvention is administered near or at the tumor site. In exemplaryaspects, the compound or composition of the invention is administeredvia injection at the tumor site.

The compounds and compositions provided herein also may be used foraffecting leukocyte function. The invention accordingly provides amethod of inhibiting leukocyte adhesion, spreading, migration, orchemotaxis. The method comprises the step of contacting a leukocyte witha compound or composition of the invention in an amount effective toinhibit leukocyte adhesion, spreading, migration, or chemotaxis. Sincethese leukocyte functions are related to inflammation, the inventionadditionally provides a method of inhibiting or treating inflammation ina subject in need thereof. The method comprises the step ofadministering to the subject a compound or composition of the inventionin an amount effective to inhibit or treat inflammation. In exemplaryembodiments, the compound or composition is administered to the subjectsystemically, e.g., parenterally (e.g., via intravenous injection).

Treatment, Prevention, and Inhibition

As used herein, the term “treat,” as well as words related thereto, donot necessarily imply 100% or complete treatment. Rather, there arevarying degrees of treatment of which one of ordinary skill in the artrecognizes as having a potential benefit or therapeutic effect. In thisrespect, the methods of treating a stroke or a heart attack or cancer orinflammation of the invention can provide any amount or any level oftreatment. Furthermore, the treatment provided by the method of theinvention may include treatment of one or more conditions or symptoms orsigns of the stroke, heart attack, cancer or inflammation, beingtreated. Also, the treatment provided by the methods of the inventionmay encompass slowing the progression of the stroke, heart attack,cancer or inflammation. For example, the methods can treat cancer byvirtue of reducing tumor or cancer growth, reducing metastasis of tumorcells, increasing cell death of tumor or cancer cells, and the like.

As used herein, the term “prevent” and words stemming therefromencompasses delaying the onset of the medical condition being prevented.In exemplary aspects, the method delays the onset of the medicalcondition by 1 day, 2 days, 4 days, 6 days, 8 days, 10 days, 15 days, 30days, two months, 4 months, 6 months, 1 year, 2 years, 4 years, or more.As used herein, the term “prevent” and words stemming therefromencompasses reducing the risk of the medical condition being prevented.In exemplary aspects, the method reduces the risk of SCD 2-fold, 5-fold,10-fold, 20-fold, 50-fold, 100-fold, or more.

As used herein, the term “inhibit” and words stemming therefrom may notbe a 100% or complete inhibition or abrogation. Rather, there arevarying degrees of inhibition of which one of ordinary skill in the artrecognizes as having a potential benefit or therapeutic effect. In thisrespect, the compounds of the invention may inhibit the bindinginteraction between a f3 integrin and a G protein α subunit to anyamount or level. In exemplary embodiments, the inhibition provided bythe methods of the invention is at least or about a 10% inhibition(e.g., at least or about a 20% inhibition, at least or about a 30%inhibition, at least or about a 40% inhibition, at least or about a 50%inhibition, at least or about a 60% inhibition, at least or about a 70%inhibition, at least or about a 80% inhibition, at least or about a 90%inhibition, at least or about a 95% inhibition, at least or about a 98%inhibition).

Cancer

The cancer treatable by the methods disclosed herein may be any cancer,e.g., any malignant growth or tumor caused by abnormal and uncontrolledcell division that may spread to other parts of the body through thelymphatic system or the blood stream. In some embodiments, the cancer isa cancer in which an integrin and a G protein α subunit are expressed onthe surface of the cells.

The cancer in some aspects is one selected from the group consisting ofacute lymphocytic cancer, acute myeloid leukemia, alveolarrhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer ofthe anus, anal canal, or anorectum, cancer of the eye, cancer of theintrahepatic bile duct, cancer of the joints, cancer of the neck,gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear,cancer of the oral cavity, cancer of the vulva, chronic lymphocyticleukemia, chronic myeloid cancer, colon cancer, esophageal cancer,cervical cancer, gastrointestinal carcinoid tumor, Hodgkin lymphoma,hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lungcancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharynxcancer, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer,peritoneum, omentum, and mesentery cancer, pharynx cancer, prostatecancer, rectal cancer, renal cancer (e.g., renal cell carcinoma (RCC)),small intestine cancer, soft tissue cancer, stomach cancer, testicularcancer, thyroid cancer, ureter cancer, and urinary bladder cancer. Inparticular aspects, the cancer is selected from the group consisting of:head and neck, ovarian, cervical, bladder and oesophageal cancers,pancreatic, gastrointestinal cancer, gastric, breast, endometrial andcolorectal cancers, hepatocellular carcinoma, glioblastoma, bladder,lung cancer, e.g., non-small cell lung cancer (NSCLC),bronchioloalveolar carcinoma.

Subjects

In some embodiments of the invention, the subject is a mammal,including, but not limited to, mammals of the order Rodentia, such asmice and hamsters, and mammals of the order Logomorpha, such as rabbits,mammals from the order Carnivora, including Felines (cats) and Canines(dogs), mammals from the order Artiodactyla, including Bovines (cows)and Swines (pigs) or of the order Perssodactyla, including Equines(horses). In some aspects, the mammals are of the order Primates,Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans andapes). In some aspects, the mammal is a human. In some aspects, thehuman is an adult aged 18 years or older. In some aspects, the human isa child aged 17 years or less.

In exemplary aspects, the subject has a medical history of taking anintegrin antagonist or an anti-platelet drug, or the subject has beenprescribed an integrin antagonist or an anti-platelet drug. In exemplaryaspects, the subject has a medical history of coronary heart disease,heart attack, angina, stroke, transient ischemic attack, peripheralartery disease, myocardial infarction, atrial fibrillation, and/orischaemic stroke. In exemplary aspects, the subject has a medicalhistory comprising angioplasty, stent placement, and/or heart bypass orvalve replacement surgery. In exemplary aspects, the subject suffersfrom acute coronary syndrome, unstable angin, or non-ST-segmentelevation myocardial infarction.

Kits

In some embodiments, the composition comprising a compound of theinvention, a pharmaceutically acceptable salt thereof, a conjugatecomprising the compound, or a multimer or dimer comprising the compound,is provided as a kit or package or unit dose. “Unit dose” is a discreteamount of a therapeutic composition dispersed in a suitable carrier.Accordingly, provided herein are kits comprising a compound of theinvention, a pharmaceutically acceptable salt thereof, a conjugatecomprising the compound, or a multimer or dimer comprising the compound.

In some embodiments, the components of the kit/unit dose are packagedwith instructions for administration to a patient. In some embodiments,the kit comprises one or more devices for administration to a patient,e.g., a needle and syringe, a dropper, a measuring spoon or cup or likedevice, an inhaler, and the like. In some aspects, the compound of theinvention, a pharmaceutically acceptable salt thereof, a conjugatecomprising the compound, or a multimer or dimer comprising the compound,is pre-packaged in a ready to use form, e.g., a syringe, an intravenousbag, an inhaler, a tablet, capsule, etc. In some aspects, the kitfurther comprises other therapeutic or diagnostic agents orpharmaceutically acceptable carriers (e.g., solvents, buffers, diluents,etc.), including any of those described herein. In particular aspects,the kit comprises a compound of the invention, a pharmaceuticallyacceptable salt thereof, a conjugate comprising the compound, or amultimer or dimer comprising the compound, along with an agent, e.g., atherapeutic agent, used in chemotherapy or radiation therapy.

The following examples are given merely to illustrate the presentinvention and not in any way to limit its scope.

EXAMPLES Example 1

The following materials and methods were used in the studies describedin Example 2.

Reagents

Myristoylated Gα₁₃ SRI peptide, Myr-LLARRPTKGIHEY (mSRI; SEQ ID NO: 46),and myristoylated-scrambled peptide (Myr-LIRYALHRPTKEG; SEQ ID NO: 47)were synthesized and purified at the Research Resource Center atUniversity of Illinois, Chicago (S1). Expression and purification ofrecombinant Gα₁₃ was described previously (S2). Anti-RhoA antibody andcell permeable C3 transferase (C3 toxin) were purchased fromCytoskeleton, Inc.; anti-Gα₁₃ (SC410), anti-c-Src (sc-18) and anti-mouseintegrin β₃ (sc-6627) antibodies were from Santa Cruz Biotechnology,Inc; anti-phospho-Src Y⁴¹⁶ antibody was obtained from Cell Signaling;anti-human integrin β₃ antibody, MAb 15 and anti-α_(IIb) β₃ antibody,D57, were kindly provided by Dr. Mark Ginsberg (University ofCalifornia, San Diego, La Jolla, Calif.); anti-GPIb monoclonal antibodyLJP3 was kindly provided by Dr. Zaverio Ruggeri, the Scripps ResearchInstitute, La, Jolla, Calif.); anti-tubulin and anti-flag antibodieswere purchased from Sigma-Aldrich; lipofectamine 2000, viraPowerlentivirus expression system, Alexa Fluor 546-conjugated phalloidin,Alexa Fluor 633-conjugated phalloidin, and Alexa Fluor 546-conjugatedanti-mouse IgG antibody were from Invitrogen; Y27632 was purchased fromCalbiochem.

Platelets Preparation, Platelet Spreading and Clot Retraction.

Studies using human platelets were approved by the Institutional ReviewBoard of University of Illinois at Chicago. Human washed platelets wereprepared from freshly drawn blood of healthy volunteers and resuspendedin modified Tyrode's buffer (12 mM NaHCO₃, 138 mM NaCl, 5.5 mM glucose,2.9 mM KCl, 2 mM MgCl₂, 0.42 mM NaH₂PO₄, 10 mM HEPES, (pH 7.4) (S3, S4).Animal studies were approved by the institutional Animal Care Committeeof University of Illinois at Chicago. Blood was freshly drawn from theinferior vena cava in isoflurane-anethetized mice. Mouse platelets wereisolated and washed using methods previously described (S4, S5). Foranalyzing platelet spreading on integrin ligand fibrinogen, washedplatelets were allowed to spread on 100 μg/ml fibrinogen-coatedcoverslips at 37° C. for 90 minutes, stained and viewed with a Leica RMIRB microscope or Zeiss LSM510 META confocal microscope as previouslydescribed (S5). Clot retraction was analyzed with the previouslydescribed method (S6, S7). Briefly, mouse platelets (6×10⁸/ml) wereresuspended in platelet-depleted human plasma, and 0.4 U/ml α-thrombinwas added to initiate coagulation. The clots were photographed atvarious time points. Sizes of retracted clots on photographs werequantified using NIH Image J software. Statistical significance wasdetermined using t-test.

Co-Immunoprecipitation and Binding Assays

Human platelets or CHO cells expressing recombinant integrin α_(IIb)β₃were solubilized in modified RIPA Buffer (50 mM Tris, pH 7.4, 10 mMMgCl₂, 150 mM NaCl, 1% NP-40, 1 mM sodium orthovanadate, 1 mM NaF), orRIPA buffer (25 mM Tris, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodiumdeoxycholate, 0.1% SDS) and complete protease inhibitor cocktail tablets(1 tablet/5 ml buffer, Roche). As previously described (S7), celllysates were incubated with 2 μg/ml of D57 (antibody to integrinα_(IIb)β₃), LJP3 (antibody against GPIb), or mouse IgG, and furtherincubated with protein G-conjugated Sepharose beads. Cell lysates werealso incubated with rabbit anti-Gα₁₃ IgG antibody (1.5 μg/ml) or anequal amount of rabbit IgG and further incubated with proteinA-conjugated Sepharose beads. After 3-6 washes with lysis buffer,immunoprecipitates were analyzed by SDS-polyacrylamide gelelectrophoresis and Western blots with antibodies against β₃ (MAb15),GPIb (anti-IbαC (S8)) or Gα₁₃. In some experiments, 1 μM GDP, 1 μM GTPγSor 30 μM AlF₄ ⁻ were added to the reaction to assess the effect of Gα₁₃activation on integrin binding. In other experiments, 250 μM mSRI orscrambled control peptide was incubated with platelet lysates prior toimmunoprecipitation. GST bead pull down analysis was previouslydescribed (S7). Purified Gα₁₃ was incubated with glutathione beads-boundto GST, GST-β₁CD or GST-β₃ CD at 4° C. overnight. Bead-bound proteinswere analyzed by immunoblotting. For GST-β₃CD cDNA construction,integrin-β₃ cytoplasmic domain (716-762) cDNA was generated by PCR andcloned into pGEX-4T2 vector using Bam HI and Xho I restriction sites.The forward primer is 5′-CGTGGATCCAAACTCCTCATCACCATCCACGACC-3′ (SEQ IDNO: 48); the reverse primer is 5′-GCGCTCGAGTTAAGTGCCCCGGTACGTGATATTG-3′(SEQ ID NO: 49). For GST-I31CD cDNA construction, β₁ cytoplasmic domain(752-798) cDNA was amplified by PCR and cloned into pGEX-4T1 vectorusing EcoRI and Xho I restriction sites. The primer sequences are: (1)forward: 5′-GCGAATTCAAGCTTTTAATGATAATTCATGAC-3′ (SEQ ID NO: 50); (2)reverse: GCGCTCGAGTCATTTTCCCTCATACTTCGGATT-3′ (SEQ ID NO: 51). GST,GST-β₁CD and GST-β₃CD were purified from BL21 (DE3) E. coli usingglutathione-conjugated beads.

Expression of Wild Type Gα₁₃ and Truncation Mutants for β₃ Binding

Human Gα₁₃ cDNA (S9) was tagged with Flag-epitope at N-terminus usingPCR with the Flag cDNA sequence incorporated into the forward primer,and then subcloned into pCDEF3 vector using Kpn I and Not I restrictionsites. The forward primer sequence is5′-GCGGGTACCGCCATGGACTACAAGGACGACGATGACAAGGCGGACTTCC-TGCCGTCGCGGTCCGT-3′(SEQ ID NO: 52). The reverse primer sequence is5′-GGCCGGCGGCCGCTCACTGTAGCATAAGCTGCTTGAGGTT-3′ (SEQ ID NO: 53).Truncation mutants of Gα₁₃ were generated using PCR with reverse primersequences5′-GGCCGGCGGCCGCTCAAATATCTTGTTGTGATGGAAT-ATAATCTGGTTCTCCAAGTTTATCCAAG-3′(SEQ ID NO: 54). for mutant 1-196; and5′-GGCCGGCGGCCGCTCATTCAAAGTCGTATTCATGGATGCC-3′ (SEQ ID NO: 55). formutant 1-212.

cDNA encoding Flag-tagged wild type Gα₁₃ or Gα₁₃ mutants weretransfected into 293FT cells using lipofectamine 2000. Cell lysates wereprepared 48 hours after transfection. Flag-tagged wild type or mutantGα₁₃ in 293FT cell lysates were incubated with glutathione bead-boundGST or GST-β₃CD at 4° C. overnight. After centrifugation and washing,bead-bound proteins were immunoblotted with anti-Flag antibody.

RhoA Activity Assay.

Platelets in modified Tyrode's buffer or adherent on immobilizedfibrinogen were lysed quickly in 0.8 ml lysis buffer (50 mM Tris, pH7.4, 10 mM MgCl₂, 500 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5%deoxycholate, 10 μg/ml each of aprotinin and leupeptin, 1 mMphenylmethylsulfonyl fluoride, and 200 μM sodium vanadate). Lysates werecleared by centrifugation at 18,000 g for 2 minutes at 4° C., and thesupernatant was incubated for 1 hour with 30 μg purified GST-RhotekinRhoA-binding domain fusion protein (GST-RBD) bound toglutathione-Sepharose beads (S10). Samples were washed three times usingwashing buffer (50 mM Tris, pH 7.4, 10 mM MgCl₂, 150 mM NaCl, 1% TritonX-100) and then immunoblotted with an anti-RhoA monoclonal antibody.Cell lysates were also immunoblotted with anti-RhoA as loading control.

Interference of Gα₁₃ Expression with siRNA, Rescue with siRNA-ResistantGα₁₃, and Bone Marrow Transplantation.

Two different Gα₁₃ siRNA target sequences were used: siRNA #1,5′-GTCCACCTTCCTGAAGCAG (SEQ ID NO: 56); siRNAi #2,5′-GGAGATCGACAAATGCCTG (SEQ ID NO: 57). Scrambled siRNA sequence is:5′-GAGGAGCCGACGCTTAATA-3′ (SEQ ID NO: 58). These sequences are conservedin hamsters and mice. Lentivirus was prepared by co-transfection ofpLL3.7-scrambled siRNA or pLL3.7-Gα₁₃ siRNA (#1 and #2) with pLP1, pLP2and pLP/VSVG plasmids (Invitrogen) into ˜90% confluent 293FT cells usingLipofectamine 2000. 48 hours after transfection, cell culture mediumcontaining virus was filtered, titered and stored at −80° C. Bone manowcells from 6-8 week old healthy C57/BL mice were isolated asepticallyfrom femurs and tibias. Stem cells were negatively selected by MACSLineage cell depletion kit (Miltenyi Biotec) and cultured in RPMI 1640complete medium with 10 ng/ml interleukin-3, 10 ng/ml interleukin-6, 10ng/ml granulocyte-macrophage colony stimulating factor (GM-CSF), and 100ng/ml stem cell factor (SCF). 50 multiplicity of infection (MOI)lenti-virus was used to infect mice bone marrow stem cells twice with 6μg/ml polybrene. 48 hours after infection, 5×10⁶ stem cells resuspendedin PBS were transplanted by retrobulbar injection into lethallyirradiated (10.5 Gy) C57/BL mice one day after irradiation (S11). ThesiRNA-resistant mutants of Gα₁₃ were generated by PCR.

These mutants changed the Gα₁₃ siRNA #1 target sequence to5′-GTCCACCTTttTaAAGCAG-3′ (SEQ ID NO: 59). or siRNA #2 target sequenceto 5′-GGAGATCGAtAAgTGCCTG-3′ (SEQ ID NO: 60). without changing the aminoacid sequence of Gα₁₃. The mutants were subcloned into pLenti6/V5-Destvector using EcoR I and Sal I restriction sites in PCR fragments andEcoRI and Xho I restriction sites in the vector. The primer sequencesare as follows: (1) Flag tagged forward primer:5′-CGGAATTCG-CCATGGACTACAAGGACGACGATGACAAGGCGGACTTCCTGCCGTCGCGGTCCGT-3′(SEQ ID NO: 61); (2) reverse primer:5′-GCCGTCGACTCACTGTAGCATAAGCTGCTTGAGGTT-3′ (SEQ ID NO: 62); (3) mutationsite forward primer for resistance to siRNA #1:5′-GTCCAAGGAGATCGATAAGTGCCTGTCTCGGGAA-3′ (SEQ ID NO: 63); (4) mutationsite reverse primer for resistance to siRNA #1:5′-TTCCCGAGACAGGCACTTATCGATCTCCTTGGAC-3′ (SEQ ID NO: 64); (5) mutationsite forward primer for resistance to siRNA #2:5′-CGGCAAGTCCACCTTTTTAAAGCAGATGCGGATC-3′ (SEQ ID NO: 65); (6): mutationsite reverse primer for resistance to siRNA #2:5′-GATCCGCATCTGCTTTAAAAAGGTGGACTTGCCG-3′ (SEQ ID NO: 66).

CHO cells expressing human α_(IIb)β₃ cells (123 cells) were transfectedwith Gα₁₃ siRNA construct with or without cotransfection of Flag-taggedsiRNA resistant-Gα₁₃ plasmid using lipofectamine 2000. After 30 hours,the cells were detached by 0.53 mM EDTA in phosphate-buffered saline,and allowed to spread on 100 μg/ml fibrinogen. For c-Srcphosphorylation, cells were solubilized in SDS-sample buffer andimmunoblotted with anti c-Src pY416 antibody. For immuno-stainingexperiments, 123 cells co-transfected with Gα₁₃ siRNA plasmid andsiRNA-resistant Gα₁₃ plasmid were allowed to adhere to 100 μg/mlfibrinogen for 1 hour, fixed by 4% paraformaldehyde, and stained byanti-flag antibody and Alexa Fluor 546-conjugated secondary antibody,and Alexa Fluor 633-conjugated phalloidin. Images were obtained using aZeiss LSM510META confocal microscope.

Quantitation and Statistics

Western blot bands were scanned, and analyzed for uncalibrated opticaldensity using NIH Image J software. Student t-test was used to determinestatistical significance.

Example 2

Integrins mediate cell adhesion and transmit signals within the cellthat lead to cell spreading, retraction, migration, and proliferation(1). Thus, integrins have pivotal roles in biological processes such asdevelopment, immunity, cancer, wound healing, hemostasis and thrombosis.The platelet integrin, α_(IIb)β₃, typically displays bidirectionalsignaling function (2, 3). Signals from within the cell activate bindingof α_(IIb)β₃ to extracellular ligands, which in turn triggers signalingwithin the cell initiated by the occupied receptor (so-called“outside-in” signaling). A major early consequence of integrin“outside-in” signaling is cell spreading, which requires activation ofthe protein kinase c-Src and c-Src-mediated inhibition of the smallguanosine triphosphatase (GTPase) RhoA (4-7). Subsequent cleavage of thec-Src binding site in β₃ by calpain allows activation of RhoA, whichstimulates cell retraction (7, 8). The molecular mechanism couplingligand-bound α_(IIb)β₃ to these signaling events has been unclear.

Heterotrimeric guanine nucleotide-binding proteins (G proteins) consistof Gα, Gβ and Gγ subunits (9). G proteins bind to the intracellular sideof G-protein coupled receptors (GPCR) and transmit signals that areimportant in many intracellular events (9-11). Gα₁₃, when activated byGPCRs, interacts with Rho guanine-nucleotide exchange factors (RhoGEF)and thus activates RhoA (11-14), facilitating contractility and roundingof discoid platelets (shape change). To determine whether Gα₁₃ functionsin signaling from ligand-occupied integrin, we investigated whetherinhibition of Gα₁₃ expression with small interfering RNA (siRNA)affected α_(IIb)β₃-dependent spreading of platelets on fibrinogen, whichis an integrin ligand. We isolated mouse bone marrow stem cells andtransfected them with lentivirus encoding Gα₁₃ siRNA. The transfectedstem cells were transplanted into irradiated C57/BL6 mice (15). Four tosix weeks after transplantation, nearly all platelets isolated fromrecipient mice were derived from transplanted stem cells as indicated bythe enhanced green fluorescent protein (EGFP) encoded in lentivirusvector (FIG. 5, FIG. 1A). Platelets from Gα₁₃ siRNA-transfected stemcell recipient mice showed >80% decrease in Gα₁₃ expression (FIG. 1B).When platelets were allowed to adhere to immobilized fibrinogen[α_(IIb)β₃ binding to immobilized fibrinogen does not require prior“inside-out” signaling activation (16)], platelets depleted of Gα₁₃spread poorly as compared with control platelets (FIG. 1A, FIG. 6). Theinhibitory effect of Gα₁₃ deficiency is unlikely to be caused by itseffect on GPCR-stimulated Gα₁₃ signaling because (i) washed restingplatelets were used and no GPCR agonists were added, and (ii) priortreatment with 1 mM aspirin [which abolishes thromboxane A₂ (TXA₂)generation (17)] did not affect platelet spreading on fibrinogen (FIG.6), making it unlikely the endogenous TXA₂-mediated stimulation of Gα₁₃.Furthermore, Gα₁₃ siRNA inhibited spreading of Chinese hamster ovary(CHO) cells expressing human α_(IIb)β₃ (123 cells) (18), which wasrescued by an siRNA-resistant Gα₁₃ (FIG. 7). Thus, Gα₁₃ appears to beimportant in integrin “outside-in” signaling leading to cell spreading.

To determine whether Gα₁₃ serves as an early signaling mechanism thatmediates integrin-induced activation of c-Src, we measuredphosphorylation of c-Src at Tyr⁴¹⁶ (which indicates activation of c-Src)in control and fibrinogen-bound cells. Depletion of Gα₁₃ in mouseplatelets or 123 cells abolished phosphorylation of c-Src Tyr⁴¹⁶ (FIG.1C, FIG. 7), indicating that Gα₁₃ may link integrin α_(IIb)β₃ and c-Srcactivation. Because c-Src inhibits RhoA (7, 19), we also tested the roleof Gα₁₃ in regulating activation of RhoA. RhoA activity was suppressedto baseline 15 minutes after platelet adhesion, and became activated at30 minutes (FIG. 1C), which is consistent with transient inhibition ofRhoA by c-Src (7). The integrin-dependent delayed activation of RhoA wasnot inhibited by depletion of Gα₁₃, indicating its independence of theGPCR-Gα₁₃-RhoGEF pathway (FIG. 1C). In contrast, depletion of Gα₁₃accelerated RhoA activation (FIG. 1C). Thus, Gα₁₃ appears to mediateinhibition of RhoA. The inhibitory effect of Gα₁₃ depletion on plateletspreading was reversed by Rho-kinase inhibitor Y27632 (FIG. 1A),suggesting that Gα₁₃-mediated inhibition of RhoA is important instimulating platelet spreading. These data are consistent with Gα₁₃mediating integrin “outside-in signaling” inducing c-Src activation,RhoA inhibition, and cell spreading.

The integrin α_(IIb)β₃ was co-immunoprecipitated by anti-Gα₁₃ antibody,but not control IgG, from platelet lysates (FIG. 2A). Conversely, anantibody to β₃ immunoprecipitated Gα₁₃ with β₃ (FIG. 2B).Co-immunoprecipitation of β₃ with Gα₁₃ was enhanced by GTP-γS or AlF₄(FIG. 2A, FIG. 8). Thus, β₃ is present in a complex with Gα₁₃,preferably the active GTP-bound Gα₁₃. To determine whether Gα₁₃ directlybinds to the integrin cytoplasmic domain, we incubated purifiedrecombinant Gα₁₃ (20) with agarose beads conjugated with glutathioneS-transferase (GST), or a GST-1β₃ cytoplasmic domain fusion protein(GST-1₃CD). Purified Gα₁₃ bound to GST-β₃CD, but not to GST (FIG. 2C).Purified Gα₁₃ also bound to the β₁ integrin cytoplasmic domain fusedwith GST (GST-β₁CD) (FIG. 2D). The binding of Gα₁₃ to GST-β₃CD andGST-β₁CD was detected with GDP-loaded Gα₁₃, but enhanced by GTP-γS andAlF₄ (FIGS. 2C, 2D), indicating that the cytoplasmic domains of β₃ andβ₁ can directly interact with Gα₁₃, and GTP enhances the interaction.The Gα₁₃-β₃ interaction was enhanced in platelets adherent tofibrinogen, and by thrombin, which stimulates GTP binding to Gα₁₃ viaGPCR (FIG. 2E). Hence, the interaction is regulated by both integrinoccupancy and GPCR signaling.

To map the β₃ binding site in Gα₁₃, we incubated cell lysates containingFlag-tagged wild type or truncation mutants of Gα₁₃ (FIG. 9) withGST-β₃CD beads. GST-β₃CD associated with wild type Gα₁₃ and the Gα₁₃1-212 fragment containing a helical region and switch region I (SRI),but not with the Gα₁₃ fragment containing residues 1-196 lacking SRI(FIG. 2F). Thus, SRI appears to be critical for β₃ binding. To furtherdetermine the importance of SRI, Gα₁₃-β₃ binding was assessed in thepresence of a myristoylated synthetic peptide, Myr-LLARRPTKGIHEY (mSRI;SEQ ID NO: 45), corresponding to the SRI sequence of Gα₁₃ (197-209)(21). The mSRI peptide, but not a myristoylated scrambled peptide,inhibited Gα₁₃ binding to β₃ (FIG. 2G), indicating that mSRI is aneffective inhibitor of β₃-Gα₁₃ interaction. Therefore, we furtherexamined whether mSRI might inhibit integrin signaling. Treatment ofplatelets with mSRI inhibited integrin-dependent phosphorylation ofc-Src Tyr⁴¹⁶ and accelerated RhoA activation (FIG. 3A). The effect ofmSRI is unlikely to result from its inhibitory effect on the binding ofRhoGEFs to Gα₁₃ SRI because Gα₁₃ binding to RhoGEFs stimulates RhoAactivation, which should be inhibited rather than promoted by mSRI (21).Thus, these data suggest that β₃-Gα₁₃ interaction mediates activation ofc-Src and inhibition of RhoA. Furthermore, mSRI inhibitedintegrin-mediated platelet spreading (FIG. 3B), and this inhibitoryeffect was reversed by C3 toxin (which catalyzes the ADP ribosylation ofRhoA) or Y27632, confirming the importance of Gα₁₃-dependent inhibitionof RhoA in platelet spreading. Thrombin promotes platelet spreading,which requires cdc42/Rac pathways (22). However, thrombin-promotedplatelet spreading was also abolished by mSRI (FIG. 3B), indicating theimportance of Gα₁₃-β₃ interaction. Thus, Gα₁₃-integrin interactionappears to be a mechanism that mediates integrin signaling to c-Src andRhoA, thus regulating cell spreading.

To further determine whether Gα₁₃ mediates inhibition ofintegrin-induced RhoA-dependent contractile signaling, we investigatedthe effects of mSRI and depletion of Gα₁₃ on platelet-dependent clotretraction (shrinking and consolidation of a blood clot requiresintegrin-dependent retraction of platelets from within) (7, 8). Clotretraction was accelerated by mSRI and depletion of Gα₁₃ (FIGS. 4, A andB, FIG. 10), indicating that Gα₁₃ negatively regulates RhoA-dependentplatelet retraction and coordinates cell spreading and retraction. Thecoordinated cell spreading-retraction process is also important in woundhealing, cell migration and proliferation (23).

The function of Gα₁₃ in mediating the integrin-dependent inhibition ofRhoA contrasts with the traditional role of Gα₁₃, which is to mediateGPCR-induced activation of RhoA. However, GPCR-mediated activation ofRhoA is transient, peaking at 1 minute after exposure of platelets tothrombin, indicating the presence of a negative regulatory signal (FIGS.4, D and F). Furthermore, thrombin-stimulated activation of RhoA occursduring platelet shape change before substantial ligand binding tointegrins (FIGS. 4, C, D and F). In contrast, following thrombinstimulation, β₃ binding to Gα₁₃ was diminished at 1 minute whenGα₁₃-dependent activation of RhoA occurs, but increased after theoccurrence of integrin-dependent platelet aggregation (FIGS. 4, E andF). Thrombin-stimulated binding of Gα₁₃ to α_(IIb)β₃ and simultaneousRhoA inhibition both require ligand occupancy of α_(IIb)β₃, and areinhibited by the integrin inhibitor RGDS (FIG. 4, D-F). Thus, our studydemonstrates not only a function of integrin α_(IIb)β₃ as anon-canonical Gα₁₃-coupled receptor but also a new concept ofGα₁₃-dependent dynamic regulation of RhoA, in which Gα₁₃ mediatesinitial GPCR-induced RhoA activation and subsequent integrin-dependentRhoA inhibition (FIG. 4G). These findings are important for ourunderstanding on how cells spread, retract, migrate, and proliferate,which is fundamental to development, cancer, immunity, wound healing,hemostasis and thrombosis.

Example 3

The following materials and methods were carried out and the results ofsome are described in Example 4.

Animals and Reagents

Integrin β₃ ^(−/−) mice were obtained from the Jackson Laboratory.Myristoylated peptides were synthesized and purified at the ResearchResource Center at University of Illinois. These peptides include: mP₁₃(Myr-KFEEERARAKWDT; SEQ ID NO: 67), mP₇ (Myr-KFEEERA) and mP₅(Myr-EEERA; SEQ ID NO: 68) and myristoylated scrambled peptide for mP₁₃(Myr-EEARERKDWAKFT; SEQ ID NO: 69); myristoylated scrambled peptide formP₇ (Myr-EAREKFE; SEQ ID NO: 70) and myristoylated scrambled peptide formP₅ (Myr-EEARE; SEQ ID NO: 71). Human integrin β₃ cDNA was cloned intopCDNA3.1 vector following digestion with Hind III and Xho I, orpLenti6-V5/Dest vector following digestion with EcoR I, Mfe I, and XhoI. Truncation mutants and integrin E to A mutants were either previouslyreported²³ or generated using PCR and cloned into pCDNA3.1 vector by BamHI and Xho I. The primer sequences used are: (1): ITGB₃-UP:5′-GCGAAGCTTGCCGCCATGGACCGAGCGCGGCCGCGGCCCCGGCCGCTCT-3′ (SEQ ID NO: 72);(2): ITGB₃-728DN: 5′-GCGCTCGAGTCAAGCGAATTCTTTTCGGTCGTGGATGGTGATGAG-3′(SEQ ID NO: 73); (3): ITGB₃-715DN:5′-GCGCTCGAGTCACCAGATGAGCAGGGCGGCAAGGCCAATGAGCAG-3′ (SEQ ID NO: 74);(4): Itgb₃-E731A-up: 5′-AAGAATTCGCTAAATTTGCAGAAGAACGCGCCAGAGCAA-3′ (SEQID NO: 75); (5): Itgb₃-E732A-up:5′-AAGAATTCGCTAAATTTGAGGCAGAACGCGCCAGAGCAA-3′ (SEQ ID NO: 76); (6):Itgb₃-E733A-up: 5′-AAGAATTCGCTAAATTTGAGGAAGCACGCGCCAGAGCAA-3′ (SEQ IDNO: 77); (7): Itgb₃-E731-733A-up:5′-AAGAATTCGCTAAATTTGCAGCAGCACGCGCCAGAGCAA-3′ (SEQ ID NO: 78); (8):Mfe-ITGB₃-Up: 5′-CCGCAATTGGCCGCCATGGACCGAGCGCGGCCGCGGCCCCGGCCGCTCT-3′(SEQ ID NO: 79); (9): Xho I-ITGB₃-DN:5′-GCGCTCGAGTTAAGTGCCCCGGTACGTGATATTG-3′ (SEQ ID NO: 80). Human integrinβ₈-CD cDNA was cloned into pGEX4T-1 vector following digestion with BamHI and Xho I. Primer sequences used are: (1): ITGB₈-UP: 5′-CGTGGATCCATTAGACAGGTGATACTACAATGG-3′ (SEQ ID NO: 81); (2): ITGB₈-Dn:5′-GCGCTCGAGTTAGA AGTTGCACCTGAAAGTTTC-3′ (SEQ ID NO: 82). GST-β₃CD andrecombinant Gα₁₃ purification was described previously⁸. Human talinhead domain cDNA corresponds to N-terminal talin amino acid residues1-433, which was cloned into pCDNA3.1 vector and pMal-C2 vector betweenEcoR I and Xho I sites. Anti-RhoA antibody was purchased fromCytoskeleton, Inc.; anti-Gα₁₃(sc410), anti-total c-Src (scl8),anti-talin (sc7534), and anti-integrin β₃ (sc6627) antibodies were fromSanta Cruz Biotechnology, Inc; anti-Gα₁₃(26004) was from NewEast;anti-phospho-Src Y⁴¹⁶ antibody was obtained from Cell Signaling;anti-talin (TA205) was from Millipore; anti-human integrin β₃ antibody,MAb 15 and 8053 rabbit serum were kindly provided by Dr. Mark Ginsberg(University of California, San Diego, La Jolla, Calif.); lipofectamine2000, viraPower lentivirus expression system, Alexa Fluor 546-conjugatedphalloidin, and Fluor 546-conjugated anti-mouse secondary antibody werefrom Invitrogen; Y-27632 is from Calbiochem.

Platelets Preparation and Spreading on Immobilized Fibrinogen

Studies using human platelets were approved by institutional reviewboard of University of Illinois at Chicago. Human washed platelets wereprepared from freshly drawn blood of healthy volunteers as previouslydescribed and resuspended in modified Tyrode's buffer (12 mM NaHCO₃, 138mM NaCl, 5.5 mM glucose, 2.6 mM KCl, 1 mM MgCl₂, 0.42 mM NaH₂PO₄, 2.5 mMHEPES, 1 mM CaCl₂ pH 7.4, 0.1% BSA)²⁴. Mice were anesthetized byisoflurane (Pharmaceutical, Inc) and platelets were prepared fromfreshly drawn blood from the inferior vena cava and washed using thepreviously described method²⁵. For analyzing platelet spreading onintegrin ligand fibrinogen, washed platelets were allowed to spread on100 μg/ml fibrinogen-coated coverslips at 37° C. for different timepoints, fixed, permeabilized, stained and viewed with a Leica RMI RBmicroscope or Zeiss LSM5 10 META confocal microscope as previouslydescribed⁸.

Fibrinogen Binding Assay

Washed human or mouse platelets resuspended in modified Tyrode's buffer(3×10⁸/ml, 50 μl) were incubated with 10 μg/ml OregonGreen⁴⁸⁸-conjugated fibrinogen (Molecular Probes) and 50 μM PAR4AP for30 minutes at 22° C. as previously described²⁶. The reaction was dilutedwith 0.5 ml of PBS and analyzed by flow cytometry using FACS Caliber (BDBiosciences, San Jose, Calif.).

Co-Immunoprecipitation and In Vitro Binding Assays

Platelets or CHO-1b9 cells expressing recombinant integrin α_(IIb)β₃^(14, 23) were solubilized in modified RIPA Buffer (50 mM Tris, pH 7.4,150 mM NaCl, 1% NP-40, 1 mM sodium orthovanadate, 1 mM NaF) withcomplete protease inhibitor cocktail tablets. Cell lysates wereincubated with rabbit anti-Gα₁₃ IgG (1.5 μg/ml), anti-integrin β₃ rabbitserum (5 μl/ml) or an equal amount of rabbit IgG or pre-immune serum at4° C. overnight, and subsequently with protein A-conjugated Sepharosebeads for 1 hour. For the integrin β₃ clustering experiment, humanplatelets (3×10⁸) were stimulated with 0.025 U/ml α-thrombin for 2minutes at room temperature (to avoid platelet aggregation) with orwithout 2 mM RGDS, then incubated with 5 μl anti-integrin β₃ 8053 rabbitserum at room temperature for 1 hour, and 5 μg goat anti-rabbitsecondary antibody for another 1 hour. Platelets were then solubilizedin modified RIPA Buffer. For the un-clustering control and pre-immuneserum control, cells were solubilized in modified RIPA Buffer first,than incubated with 5 μl 8053 serum or pre-immune serum. After 3-6washes with lysis buffer, immunoprecipitates were analyzed bySDS-polyacrylamide gel electrophoresis and Western blots usingantibodies against β₃, talin, or Gα₁₃. In some experiments, 500 μMcontrol or inhibitor peptides, mP₅, mP₇ and mP₁₃ were incubated withplatelet lysates prior to immunoprecipitation, or 2 mM RGDS was addedinto washed human platelets before stimulation by 0.025 U/ml α-thrombin.GST bead pull down analysis was previously described⁸. Purified Gα₁₃ orMBP-talin head were incubated with glutathione beads-bound to GST orGST-β₃CD at 4° C. overnight. Bead-bound proteins were analyzed byimmunoblotting.

RhoA Activity Assay

Platelets or α_(IIb)β₃-expressing CHO cells in modified Tyrode's bufferor adherent on immobilized fibrinogen were solubilized in 0.8 ml lysisbuffer (50 mM Tris, pH 7.4, 10 mM MgCl₂, 500 mM Nacl, 1% Triton X-100,0.1% SDS, 0.5% deoxycholate, 10 μg/ml each of aprotinin and leupeptin, 1mM phenylmethylsulfonyl fluoride, and 200 μM sodium vanadate). Lysateswere cleared at 14,000 rpm for 2 minutes at 4° C., and the supernatantwas incubated for 1 hour with 30 μg purified GST-Rhotekin RhoA-bindingdomain fusion protein (GST-RBD) bound to glutathione-Sepharose beads aspreviously described²⁷. Samples were washed three times with 50 mM Tris,pH 7.4, 10 mM MgCl₂, 150 mM NaCl, 1% Triton X-100, and thenimmunoblotted with an anti-RhoA monoclonal antibody. Cell lysates werealso immunoblotted with anti-RhoA as loading control.

Bone Marrow Transplantation

Lenti-virus was prepared by co-transfection of pLenti6/V5-Dest vectorinserted with integrin β₃ or AAA mutant β₃ cDNA with pLP1, pLP2 andpLP/VSVG plasmids (Invitrogen) into ˜90% confluent 293FT cells usingLipofectamine 2000. 48-72 hours after transfection, cell culture mediumcontaining virus was concentrated, titered and stored at −80° C. Bonemarrow cells from 6-8 week old integrin β₃ ^(−/−) mice (JacksonLaboratories) were isolated aseptically from femurs and tibias. Stemcells were negatively selected by MACS Lineage cell depletion kit(Miltenyi Biotech) and cultured in RPMI 1640 complete medium with 10ng/ml interleukin-3, 10 ng/ml interleukin-6, 10 ng/mlgranulocyte-macrophage colony stimulating factor (GM-CSF), and 100 ng/mlstem cell factor (SCF). 50 multiplicity of infection (MOI) lenti-viruswas used to infect mice bone marrow stem cells twice with 6 μg/mlpolybrene. 48 hours after infection, 5×10⁶ stem cells resuspended in PBSwere transplanted by retrobulbar injection into irradiated (5 Gy)integrin β₃ ^(−/−) mice one day after irradiation⁸.

Immunofluorescence and Confocal Microscopy

Coverslides were pre-coated with 100 μg/ml fibrinogen and blocked with5% BSA in PBS. 300 μl α_(IIb)β₃-expressing CHO cells (expressing WT ormutant integrins; 1×10⁵/ml) or platelets (1×10⁷/ml) suspended inTyrode's buffer were added to coverslides and incubated at 37° C. forvarious lengths of time. Cells were fixed with 4% paraformaldehyde (PFA)for 10 minutes and permeabilized with 0.1% Triton X-100 in PBS for 2minutes. After blocking with 5% BSA for 10 minutes, coverslides wereincubated with 0.2 μg/ml mAb 15, or Alexa Fluor-546 conjugatedphalloidin. For mouse platelets re-express wild type β₃ or the AAAmutant of β₃, β₃ was immuno-stained with the anti-β₃ monoclonal antibodyMAb 15 and Alexa Fluor-546 conjugated phalloidin. The slides werescanned with a Zeiss LSM5 10 META confocal microscope as previouslydescribed¹¹

Quantitation and Statistics

Image J software was used for quantitation of uncalibrated opticaldensity of Western blot bands. Paired t-test was used for statisticanalysis (mean±SD).

Example 4

As shown in the preceding examples, a G protein subunit, Gα₁₃, directlybinds to the cytoplasmic domain of β₃, and is required for integrinoutside-in signaling leading to c-Src activation, RhoA inhibition, andcell spreading⁸. To map the Gα₁₃ binding sites, we characterized thebinding of Gα₁₃ to a panel of β₃ C-terminal truncation mutants that arestably co-expressed with wild type am, in Chinese hamster ovary (CHO)cells¹⁴ (FIG. 11A). Gα₁₃ binds to β₃ deletion mutants Δ759 and Δ741 andwild type α_(IIb)β₃. However, Gα₁₃ failed to bind to the deletionmutations Δ728 and Δ715 (FIG. 11B), indicating that the β₃ sequencebetween amino acid residues K⁷²⁹ and T⁷⁴¹ is required for Gα₁₃ binding.

To determine the amino acid residues in this region in binding to Gα₁₃,we made β₃ mutants changing glutamic acid residues E731, E732 or E733 toalanine (E731A, E732A and E733A) (FIGS. 11 A and C). We also made amutant in which all these three glutamic acid residues were changed toalanine residues (AAA). These mutants were co-expressed with α_(IIb) inCHO cells, and co-immunoprecipitated with Gα₁₃ (FIG. 11C). Wild-typeintegrin β₃, but not E731A, E733A, or AAA mutants, bind to Gα₁₃ (FIG.11C), indicating that the E731 and E733 residues within the region areimportant for Gα₁₃ binding. Alignment of the sequences of differentintegrin β subunits reveals an ExE motif conserved among most β subunits(β1-β7, in β5, the first glutamic acid is replaced with aglutamine)(FIG. 11A). In our experiments, the β subunits containing theExE motif (β₁, β₂, and β₃) all interacted with Gα₁₃, indicating aconserved ExE motif in the cytoplasmic domains of most integrin βsubunits that is critical for Gα₁₃ binding. Interestingly, the Gα₁₃binding-deficient mutant of β₃, AAA, did not negatively affect thebinding of talin head domain. Thus, the ExE motif is not required fortalin binding. Therefore, we further investigated the effect of AAAmutation on Gα₁₃-dependent outside-in signaling of β₃. Wild type β₃ andthe AAA mutant β₃ in lenti-virus vectors were transfected into bonemarrow stem cells isolated from β₃ ^(−/−) mice. The transfected bonemarrow stem cells were transplanted into β₃ ^(−/−) mice following highdose irradiation. Flow cytometric analysis showed that platelets fromthe recipient mice express similar levels of wild type or AAA mutant β₃(FIG. 11E). When the platelets were plated on the integrin ligandfibrinogen, most wild type β₃-expressing platelets spread, compared tothe β₃ ^(−/−) platelets that do not spread. In contrast to plateletsexpressing wild type β₃, the spreading of AAA mutant platelets onfibrinogen was diminished (FIG. 11D). Similarly, CHO cells expressingamu/AAA mutant β₃ were also defective in spreading on fibrinogencompared with wild type α_(IIb)β₃ expressing cells (FIG. 12A). CHO cellsexpressing the β₃ AAA mutant also showed a defect in theintegrin-dependent activation of c-Src, as shown by phosphorylation atTyr⁴¹⁶, and abolished the integrin-dependent early-phase transientinhibition of RhoA during cell spreading (FIGS. 12B and C). These dataindicate that the disruption of the Gα₁₃-binding ExE motif in β₃ causeddefects in c-Src-dependent integrin outside-in signaling.

To further develop the potential competitive inhibitors of Gα13 bindingto β₃, we synthesized three myristoylated peptides mirroring sequenceswithin the K⁷²⁹-T⁷⁴¹ region of β₃: mP₁₃ (Myr-KFEEERARAKWDT (SEQ ID NO:67)), mP₇ (Myr-KFEEERA (SEQ ID NO: 68)), and mP₅ (Myr-EEERA(SEQ ID NO:413)) (FIG. 11A). These peptides were incubated with platelet lysatesbefore co-immunoprecipitation of Gα₁₃ with β₃. All 3 peptides inhibitedco-immunoprecipitation between Gα₁₃ and β₃ (FIG. 13A), showing mP₅),indicating that the sequence EEERA (SEQ ID NO: 21) is a critical Gα₁₃binding site, and these synthetic peptides are novel inhibitors ofGα₁₃-integrin interaction.

In order to determine whether these peptide inhibitors interfere withintegrin outside-in signaling, we tested the effects of thesemyristoylated β₃ cytoplasmic domain peptide on integrin outside-insignaling. Treatment of platelets with mP₅ (and mP₇ and mP₁₃, similardata not shown) also inhibited integrin-dependent c-Src activation andc-Src-dependent transient RhoA inhibition (FIG. 13 B), and alsoabolished platelet spreading on fibrinogen (FIG. 13C). The inhibitoryeffect of mP₅ on platelet spreading was reversed by the Rho kinaseinhibitor, Y27632 (FIG. 13C), suggesting that mP₅ inhibited plateletspreading mainly by blocking the Gα₁₃- and c-Src-dependent RhoAinhibitory signaling pathway, as characterized in our recent study⁸.Furthermore, mP₅ inhibited platelet aggregation induced by thrombin(FIG. 13E). In contrast, mP₅ had no effect on agonist (thrombin receptorPAR4 agonist)-induced fibrinogen binding to platelets (FIG. 13D). Thus,the inhibitor peptide mP₅ inhibited Gα₁₃-dependent integrin outside-insignaling without affecting inside-out signaling. The peptide mP₁₃ alsoinhibited integrin outside-in signaling (FIG. 14A), but alsosignificantly affected inside-out signaling as indicated by reducedfibrinogen binding (FIG. 14B), which is consistent with the previousobservations that some residues (particularly F⁷³⁰, and W⁷³⁹) in thispeptide are critical for talin interaction. These results demonstratethe selectively inhibition of integrin outside-in signaling by themembrane-permeable peptide inhibitors of Gα13-integrin interaction.These data also demonstrate that these inhibitors also inhibit plateletspreading and aggregation and therefore useful in treating thrombosis.Such selective inhibitors may allow platelet adhesion without dramaticamplification effect of outside-in signaling, and thus are potentiallyuseful as anti-thrombotics without profound bleeding side effect,compared to currently used integrin inhibitors.

Example 5

Platelet aggregation assays were carried out as follows: Plateletaggregation and secretion was measured in a turbidometric plateletaggregometer (Chronolog) at 37° C. with stirring (1000 rpm). Washedplatelets (3×10⁸/ml) in modified Tyrode's buffer were stimulated withthrombin (Enzyme Research Laboratories). For talin knockdown plateletaggregation assay stimulated with manganese and ADP, manganese and ADPwas mixed prior to experiment to achieve final concentration of 1 mMmanganese and 5 μM ADP in reaction tube. Aggregation traces shown arerepresentative of at least three independent experiments.

Example 6

Platelet Adhesion Assays were carried out as follows: As describedbefore(6), microtiter wells were coated with 30 μg/ml fibrinogen in PBSovernight. Washed human platelets in modified Tyrode's buffer in theabsence or presence of 1 mM MnCl₂ were incubated in the microtiter wellsfor one hour at 37° C. in a CO₂ incubator. After 3 washes, 50 μl ofreaction buffer (0.3% p-nitrophenyl phosphate (Sigma) in 1%Triton-X-100, 50 mM sodium acetate, pH 5.0) was added into eachmicrotiter well and incubated at 37° C. for one hour. The reaction wasstopped by adding 50 μl of 1 M NaOH. Results were determined by readingOD at 405 nm wave length. The percentage of platelet adhesion wasestimated from the ratio of the readings of adherent platelets to thatof total platelets. Statistic significance was determined using t test(n=3).

Example 7

Clot Retraction Assays were carried out as follows: Similar as describedbefore(2, 3), freshly prepared human whole blood was citrated with 1/10volume of 3.8% sodium citrate. After centrifugation at 1300 rpm for 22min with break, platelet rich plasma (PRP) was collected. Pre-incubatedof PRP with 0.05% DMSO (vehicle), 250 μM mP5 peptide, 250 μM mP13peptide, or their corresponding scrambled control peptides mP5Scr ormP13Scr for 5 minutes at room temperature. After that, 0.5 U/ml thrombinwas added into PRP and mixed gently. The clots were formed and allowedto retract at 37° C. incubator with CO₂ and were photographed at varioustimes. The two-dimensional sizes of retracted clots on photographs werequantified using Image J software and were expressed as clot size.Statistical significance was determined using a t test (n=3).

Example 8

Myristoylated peptides were synthesized: mP₅ (Myr-EEERA (SEQ ID NO:413)) and mP₁₃ (Myr-KFEEERARAKWDT (SEQ ID NO: 67)). Control peptidescomprising the scrambled sequence mP5 and mP13 were also made. Thepeptides were tested for inhibiting binding between in Gα₁₃ and β₃ andfor inhibiting binding between talin and β₃. Both peptides inhibitedco-immunoprecipitation between Gα₁₃ and β₃ (FIG. 21A), indicating thatthe minimal sequence EEERA (SEQ ID NO: 21), which includes the ExEmotif, is sufficient to bind Gα₁₃. In contrast, only mP₁₃, but not mP₅,inhibited talin association with β3, which is consistent with thepreviously data that the sequence in mP₁₃ contains importanttalin-interacting residues(4, 17, 18, 21), and indicate that the EEERA(SEQ ID NO: 21) sequence is not sufficient to interact with talin.

The peptides were also tested for inhibition of platelet spreading onfibrinogen and for inhibition of platelet adhesion to immobilizedfibrinogen. The mP₅ peptide inhibited platelet spreading on fibrinogen(FIG. 21C). The inhibitory effect of mP₅ on platelet spreading wasreversed by the Rho kinase inhibitor, Y27632 (FIG. 21C), suggesting thatmP₅ inhibited platelet spreading mainly by blocking the Gα₁₃- andc-Src-dependent RhoA inhibitory signaling pathway as characterized inour recent study(8). In contrast, mP₅ had no effect on agonist-inducedfibrinogen binding to platelets (FIG. 21B) nor does it affect plateletadhesion to immobilized fibrinogen (FIG. 21D).

The peptides were furthermore analyzed for their ability to inhibit clotretraction mediated by platelets. mP5 did not inhibit but ratheraccelerated integrin-dependent clot retraction mediated by platelets,which requires late phase outside-in signaling.

These data indicate that the β₃-based Gα₁₃ inhibitor peptide mP₅selectively inhibited the early phase of outside-in signaling withoutaffecting talin-dependent inside-out signaling or ligand-inducedintegrin activation. Nor did it inhibit the late phase outside-insignaling associated with the second wave of talin binding. Thus, Gα₁₃plays a selective role in the early phase of outside-in signaling duringwhich it binds to β₃. In contrast to mP₅, mP₁₃ not only inhibited earlyphase outside-in signaling (FIG. 21C), but also inhibited inside-outsignaling as indicated by diminished fibrinogen binding (FIG. 21B). Thispeptide also inhibited platelet adhesion to immobilized fibrinogen (FIG.21D). Furthermore, mP13 inhibited clot retraction and this inhibitionwas not reversed by manganese.

Example 9

The effect of mP5 (Myr-EEERA (SEQ ID NO:413) on platelet aggregation andsecretion was tested. As shown in FIG. 24, mP5 inhibited plateletgranule secretion and the second wave of platelet aggregation. Theseresults support the notion that mP5 selectively inhibits outside-insignaling.

Example 10

This example demonstrates the design of modified forms of the mP5peptide (Myr-EEERA; SEQ ID NO: 413). A first set of modified forms ofthe mP5 peptide are made wherein each peptide of the set retains the EEEmotif, but adds 1, 2, 3, 4, 5, or more flanking residues N-terminal tothe EEE motif or C-terminal to the EEE motif. The flanking residues arebased on the flanking sequences that naturally occur in the β₃ integrinsequence, or other beta integrin sequences. The modified peptidesinclude, for example, KFEEE (SEQ ID NO: 19), FEEER (SEQ ID NO: 84),AKFEEE (SEQ ID NO: 85), KFEEER (SEQ ID NO: 86), FEEERA (SEQ ID NO: 87),EEERAR (SEQ ID NO: 88), EEERARA (SEQ ID NO: 89), and EEERARAK (SEQ IDNO: 90). For each modified peptide synthesized, two control peptides aresynthesized: (1) a scrambled peptide having the same amino acidcomposition but having a different amino acid sequence, and (2) a lossof function peptide in which the sequence is identical except for eachof the ExE residues are changed to alanine. Each peptide is tested forthe ability to inhibit talin binding or until the affinity of thepeptide peaks.

In a second set of peptides, the peptides of the first set are modifiedto contain the second glutamic acid in the EEE motif to another aminoacid. Some will be changed to EAE or EKE. These peptides aresubsequently tested for their affinity for Gα13.

A third set of modified forms of the mP5 peptide are made wherein thepeptide is cyclized. Cyclization can increase the efficiency of deliveryand minimize extracellular cleavage of the peptide. In an exemplaryinstance, the peptides are made with a Cys at each termini (N- andC-termini) and reacted under conditions to form a disulfide bridge. Thecyclic peptides are subsequently tested for inhibitory effect onGα13-integrin interaction, biochemical markers of integrin outside-insignaling, platelet adhesion, platelet aggregation, and thrombusformation in vitro.

The above sets of peptides are tested for their ability to inhibitGα13-integrin interaction by an in vitro binding assay using purifiedGα13 and purified integrin β3 cytoplasmic domain [¹⁴]. The recombinant33 cytoplasmic domain-GST fusion protein and control GST protein areimmobilized to glutathione-beads. The beads are mixed with recombinantGα13 in the presence or absence of GTPγS. After washes, bound Gα13 isanalyzed by western blot. The modified peptides as well as each of thecontrol peptides are added to the reaction in order to determine theinhibitory effects of these peptides.

Modified forms of mP5 are additionally screened by an assay in whichmodified or control peptides are added to microtiter wells to which therecombinant 33 cytoplasmic domain is immobilized. After addition of thepeptide to each well, biotinylated Gα13 is added to each well.HRP-labeled streptoavidin in an ELISA assay is used to determine whichpeptides inhibited the interaction between Gα13 and the immobilized 33cytoplasmic domain, as those wells containing HRP-labeled streptoavidinbound to biotinylated Gα13 indicates that the peptide successfullyinhibited the binding between Gα13 and the immobilized 33 cytoplasmicdomain.

Screening of ExE peptides are additionally tested by constructing aphage library expressing the ExE motif peptides with random flankingamino acid residues, and screen for high affinity binding usingmicrotiter wells coated with Gα13 protein. The phage clones with highaffinity binding sequences are sequenced and the corresponding peptideare synthesized for further testing, as described herein.

Top performing peptides are selected and used for in vivo studies, asdescribed in the following examples.

Example 11

Micelles are nano-sized particles formed by aggregation of amphiphilicmolecules in water (FIG. 25). Peptides of the invention attached to afatty acid may be used to formulate the peptides into micelles. The ExEmotif peptide of SEQ ID NO: 87 was made into a micellar formulation, asfollows:1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000] (PEG₂₀₀₀-DSPE; Northern Lipids Inc., Vancouver, BC),L-α-phosphatidylcholine (egg PC, Type XI-E, Sigma-Aldrich, St. Louis,Mo.) and peptide was mixed at a molar ratio of 45:5:1. Micelles wereprepared using a film rehydration method, as previously described [21].The lipid film was rehydrated with 10 mM isotonic HEPES buffer (HEPES 10mM, NaCl 135 mM, pH 7.4) to form the micelle colloid. The mixture of thelipids and peptides was dissolved in methanol and chloroform, followedby evaporation using a rotary-evaporator R-215 (40 mbar, 45° C., Buchi,New Castle, Del.) to form a thin lipid layer. The lipid film wasdesiccated under vacuum (in the dark) overnight and then rehydrated with10 mM isotonic HEPES buffer (HEPES 10 mM, NaCl 135 mM, pH 7.4) to formmicelle colloid.

In a preliminary dose-dependent study, the micellar formulation ofFEEERA (SEQ ID NO: 87) was compared to the same peptide dissolved inDMSO. Whereas 250 μM of this peptide dissolved in DMSO was required forthe maximal effect on in vitro platelet aggregation, only 4 μM of thispeptide was required for the maximal effect when made into a micellarformulation (FIG. 29).

Example 12

The following experiments are performed ex vivo using isolatedplatelets, platelet rich plasma (PRP), or whole blood from human donorsand animals.

Platelet Aggregation and Granule Secretion

Blood is drawn from either human donors or from the vena cava ofanethetized mice. For preparation of washed platelets, acid citratedextrose (ACD; 85 mM trisodium citrate, 83 mM dextrose, and 21 mM citricacid) is used as anti-coagulant. For PRP, 3.2% sodium citrate is used asanti-coagulant. Either isolated platelets or PRP is preincubated withincreasing concentrations of micellar ExE motif peptides or controlscrambled peptides, and, after adding luciferase/luciferin reagents, thepeptides are tested for platelet aggregation and simultaneously recordedfor secretion of ATP from dense granules using a Lumi-aggregometer(Chronolog). Adenosine triphosphate (ADP), thrombin, PAR4AP, PARIAP,U46619, ristocetin/botrocetin and collagen are used to stimulateplatelets. P-selectin exposure, which is an indicator of α-granulesecretion, is measured using flow cytometry using anti-p-selectinantibodies^([22]).

Isolated platelets or PRP were pre-incubated with various concentrationsof either a peptide of FEEERA (SEQ ID NO: 87), or a scrambled controlthereof, dissolved in DMSO. 0.1 U thrombin was then added to the cellsto stimulate ATP secretion. ATP secretion by the cells was then measuredusing a lumi-aggregometer. % secretion relative to the scrambled controlpeptide is shown in FIG. 27.

Example 13

Platelet Adhesion and Thrombus Formation Under Flow Conditions

The effect of micellar ExE motif peptides, such as mP5 and the peptideof FEEERA (SEQ ID NO: 87), on platelet adhesion and thrombus formationare tested under flow conditions ex vivo under flow conditions thatmimic arterial blood flow. Two different types of flow adhesion assaysare set up in the laboratory using: (1) a laminar flow chamber and (2) acone-plate rheometer. Laminar flow chambers are coated withsubendothelial matrix proteins, such as collagen, von Willebrand Factor(VWF) or both. Platelets are labeled with a fluorescent dye (mepacrineor 5-chloromethylfluorescein diacetate (CMFDA)), and anti-coagulatedblood, PRP or isolated platelets are infused into the chamber at definedflow shear rates with a syringe pump. Adhesion and the formation ofplatelet-rich thrombi are monitored using an inverted fluorescencemicroscope and charge coupled device (CCD) camera. In some experiments,the thrombus size is quantitated using a confocal microscope (similar tothat described below). When using the cone-plate rheometer, glass slidesare coated with VWF or collagen, and placed on the stage of aThermo-Haake rheometer with constant temperature control.Mepacrine-labeled platelets, treated with ExE peptide micelles orcontrol micelles, are added to the glass plate and subjected to constantshear stress. Stable platelet adhesion and the formation of plateletaggregates are photographed and analyzed under a fluorescencemicroscope. Different doses of ExE motif peptides or control peptidesare incubated with platelets to obtain an inhibition constant (Ki).

Example 14

PFA-100 Analysis of ExE Motif Peptides

PFA-100 is a clinically used test of platelet function that allowspassage of blood through a cartridge coated with plateletagonists/adhesive proteins (such as epinephrine/collagen) until the timeof thrombotic occlusion occurs in the cartridge^([27]). ExE motifpeptides like mP5 and the peptide of FEEERA SEQ ID NO: 87) on thrombosisare tested in this apparatus. Blood from healthy donors who have nottaken platelet inhibitors for two weeks is anticoagulated with 3.2%sodium citrate, and micellar ExE motif peptides or control peptides areadded. The blood samples are then analyzed by PFA-100 assay asessentially described in [27].

Example 15

In Vivo Bleeding Time Analysis for Hemostatic Function

Bleeding time analysis is an indicator of overall in vivo hemostaticfunction. The ligand-binding function of integrin is critical forprimary platelet adhesion and aggregation and thus important forhemostasis. Bleeding time analysis is carried out as follows: C57BL/6mice are retro-orbitally injected with micelle formulated ExE motifpeptides (such as mP5) and scrambled controls. Distal portions of themouse tail (5 mm) are amputated with a scalpel and the tail immersed in0.15 M NaCl at 37° C. as previously described^([28]). Time to cessationof bleeding is recorded. If the bleeding fails to stop after 900seconds, the assay is stopped and pressure is applied to the tail toprevent excessive loss of blood. The assay is performed in adouble-blinded fashion. Similarly, bleeding time of wild type micetreated with current integrin antagonists, reopro and integrillin, arealso tested in comparison with the ExE motif peptides. Integrillin wastested in this fashion (with a saline control and 4 mice per group). Theresults are shown in FIG. 26 with the median value indicated. Bleedingtime for mice treated with integrillin exceeded the upper limit of theassay (900 s). Consequently, the assay was terminated and bleeding wasstopped via pressure application.

Because the ExE motif peptides do not affect the ligand binding functionof integrin αIIbβ3, it is expected that the effect of these peptides onbleeding time should be significantly reduced as compared to currentintegrin antagonists, which abolish ligand binding to integrins.

Example 16

Safety of the Micellar ExE Motif Peptides for In Vivo Use

The maximal tolerated doses (MTD) in C57BL mice are determined in orderto assess acute toxicity of the ExE motif peptide. An initial dose of 5×the maximal effective dose (as determined by ex vivo study) is injectedinto 2 mice via tail vein. A lower dose is administered if mice die orshow clinical sign of intolerance. Conversely, if none die or show signof intolerance, a higher dose is administrated. This process is repeateduntil the MTD is approached. MTD is tested in additional mice for twoweeks (5 mice of each gender). It is possible that mice tolerate thesemicellar peptides well even at high doses well above the effective dose.If this is the case, the peptides are considered safe for in vivo use.

Example 17

The effects of varying doses of the ExE motif peptides (e.g., mP5, thepeptide of FEEERA (SEQ ID NO: 87) on in vivo thrombosis are tested usingthe FeCl₃— and laser-induced injury models. Existing integrinantagonists and other anti-platelet drugs (cyclooxygenase inhibitors andP2Y12 inhibitors) are run so that the effects of the EXE motif peptidescan be compared to the effects induced by the existing drugs. Negativecontrols include vehicle controls. Additional tests are performed to seeif the use of the ExE motif peptides in combination with one or moreexisting integrin antagonists or anti-platelet drugs produce an additiveor synergistic effect on thrombosis.

Ferric Chloride-Induced Thrombosis Model

The ferric chloride injury-induced carotid artery thrombosis model hasbeen carried out in C57BL/6 mice^([29, 30]). FeCl₃-induced thrombosis iswidely used to reflect the role of platelets in the formation ofocclusive arterial thrombosis^([31]). In this study, adult mice areanesthetized by intraperitoneal injection of pentobarbital (120 mg/kg).The left common carotid artery is surgically exposed and a miniatureDoppler flow probe (Model 0.5VB; Transonic Systems, Ithaca, N.Y.) isplaced on the surface of the artery. After adding 0.9% sodium chloridesolution in the surgical wound to allow Doppler monitoring, baselineblood flow is recorded using a Transonic flowmeter. Thereafter, sodiumchloride solution is removed and filter paper (round, 1.0 mm indiameter) saturated with 10% FeCl₃ is applied to the surface of thecarotid artery immediately proximal to the flow probe. After 3 minutes,the filter paper is removed, saline solution is again administered tothe wound, and carotid blood flow is monitored. If occlusive thrombosisoccurs to the injured carotid artery, blood flow is reduced to zero. ExEmotif peptide micelles and controls are injected i.v. via the tail veinin C57BL/6 mice, then tested for occlusive thrombosis using this model.

T This assay was carried out with two EXE peptides: FEEERA (SEQ ID NO:87) and the scrambled control thereof (ERAFEE; SEQ ID NO: 91). As shownin FIG. 28, the time to occlusion was increased when the mice were givenFEEERA (SEQ ID NO: 87) as compared to its scrambled control.

Laser-Induced Injury Model of In Vivo Thrombosis Using IntravitalMicroscopy

The effects of the ExE motif peptides on in vivo thrombosis are examinedusing a laser-ablation wide-field confocal microscope system^([32]).Using this system, in vivo thrombosis in small blood vessels areobserved in real time. Using video photography, the kinetics ofthrombosis are recorded and analyzed. Fluorescently labeledplatelet-specific antibodies are injected to monitor the incorporationof platelets in thrombi. Control or ExE motif peptide-injected mice(male, 6-8 weeks) are anesthetized via intraperitoneal injection ofketamine and xylazine. A cannula is placed in the jugular vein forinjection of drugs. The cremaster muscle is exteriorized by removingconnective tissues. The muscle is fixed as a single sheet on a glassslide on an intravital microscopy tray. Rat anti-mouse CD42b antibodyconjugated with Dylight 649 is infused through the jugular cannula inmice for 5 minutes. Platelet thrombus formation is visualized using anOlympus fluorescence microscope with a 60× water immersion objectivelens and recorded using a high speed digital camera. Vaso-occlusivestate is determined by monitoring red cell flow velocity. It is expectedthat the ExE motifpeptide-treated mice show a significant reduction inocclusive thrombus formation, but minimally affect initial plateletadhesion in response to laser-induced injury.

Example 18

The Effect of In Vivo Injection of Micellar ExE Motif Peptides on ExVivo Platelet Function

Micelles containing the ExE motif peptides are injected through the tailvein of mice, and after a defined time (such as 5 min, 10 min and 30min), the mice are anesthetized and their blood drawn. Platelet functionis tested in vitro as described herein. These experiments allow thedetermination of whether injection of ExE motif peptides have an effecton overall platelet function.

Example 19

Platelet aggregation assays were performed with myristoylated peptidemP5, myristoylated peptide of SEQ: ID NO: 87, or their respectivescarmbled control peptides, as essentially described herein. Briefly,washed human platelets were preincubated with 25, 50, 100, 250, or 500μM peptide solubilized in DMSO. The platelets were subsequently inducedwith 0.09 U/ml thrombin and platelet aggregation was measured in aturbidometric platelet aggregometer. FIG. 30A provides a table of scoresfor each test peptide (mP5, myristoylated peptide of SEQ ID NO: 87) atthe indicated dose, wherein the score indicates how well the peptideinhibited platelet aggregation. As shown in FIG. 30A, the myristoylatedpeptide of SEQ ID NO: 87 was able to inhibit platelet aggregation atlower doses, as compared to the mP5 peptide. The aggregation traces foreach test peptide at 250 μM are shown in FIG. 30B.

REFERENCES

The following represents a listing of the references cited in EXAMPLES 1and 2.

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The following represents a listing of the references cited in EXAMPLES 3and 4.

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The following represents a listing of the references cited in EXAMPLE5-7.

-   1. Xi et al., Journal of Cell Biology 162, 329 (Jul. 21, 2003);-   2. Gong et al., Science 327, 340 (2010);-   3. Flevaris et al., J Cell Biol 179, 553 (Nov. 5, 2007);-   4. Yin et al., Blood 111, 658 (Jan. 15, 2008);-   5. Su et al., Blood 112, 592 (Aug. 1, 2008);-   6. Gu, et al., J Cell Biol 147, 1085 (Nov. 29, 1999);-   7. Ren and Schwartz, Methods Enzymol 325, 264 (2000); and-   8. Edelstein et al., Curr Protoc Mol Biol Chapter 14, Unitl4 20    (October, 2010).

The following represents a listing of the references cited in EXAMPLE 8.

-   1. R. O. Hynes, Cell 110, 673 (Sep. 20, 2002).-   2. S. J. Shattil, P. J. Newman, Blood 104, 1606 (Sep. 15, 2004).-   3. K. Moissoglu, M. A. Schwartz, Biol Cell 98, 547 (September,    2006).-   4. S. Tadokoro et al., Science 302, 103 (Oct. 3, 2003).-   5. M. Moser, B. Nieswandt, S. Ussar, M. Pozgajova, R. Fassler, Nat    Med 14, 325 (March, 2008).-   6. F. Ye, C. Kim, M. H. Ginsberg, J Thromb Haemost 9 Suppl 1, 20    (July, 2011).-   7. X. P. Du et al., Cell 65, 409 (May 3, 1991).-   8. H. Gong et al., Science 327, 340 (2010, 2010).-   9. A. Obergfell et al., J Cell Biol 157, 265 (Apr. 15, 2002).-   10. E. G. Arias-Salgado et al., Proc NatlAcad Sci USA 100, 13298    (Nov. 11, 2003).-   11. P. Flevaris et al., J Cell Biol 179, 553 (Nov. 5, 2007).-   12. G. Giannone, G. Jiang, D. H. Sutton, D. R. Critchley, M. P.    Sheetz, Journal of Cell Biology 163, 409 (Oct. 27, 2003).-   13. J. D. Humphries et al., Journal of Cell Biology 179, 1043 (Dec.    3, 2007).-   14. B. Nieves et al., J Cell Sci 123, 1216 (Apr. 15, 2010).-   15. X. D. Xi, R. J. Bodnar, Z. Y. Li, S. C. T. Lam, X. P. Du,    Journal of Cell Biology 162, 329 (Jul. 21, 2003).-   16. S. Patil et al., J Biol Chem 274, 28575 (Oct. 1, 1999).-   17. K. L. Wegener et al., Cell 128, 171 (Jan. 12, 2007).-   18. B. G. Petrich et al., Journal of Experimental Medicine 204, 3103    (Dec. 24, 2007).-   19. B. S. Coller, Blood 55, 169 (1980).-   20. T. P. Ugarova et al., J Biol Chem 268, 21080 (Oct. 5, 1993).-   21. E. Goksoy et al., Molecular Cell 31, 124 (Jul. 11, 2008).-   22. J. R. Haling, S. J. Monkley, D. R. Critchley, B. G. Petrich,    Blood 117, 1719 (Feb. 3, 2011).

The following represents a listing of the references cited in EXAMPLES9-18.

-   1. Roger et al., Circulation 125(1):188-197 (2012);-   2. Ruggeri, Nat Med. 8(11): 1227-1234 (2002);-   3. Shattil and Newman, Blood 104(6):1606-1615 (2004);-   4. Li et al., Arterioscler Thromb Vasc Biol. 30(12):2341-2349    (2010);-   5. Coller, Thromb Haemost. 86(1):427-443 (2001);-   6. Dyke, American heart journal 138(4 Pt 2):307-316 (1999);-   7. Saab et al., Expert opinion on drug safety 11(2):315-324 (2012);-   8. Shattil et al., Blood 91(8):2645-2657 (1998);-   9. Calderwood et al., J Biol Chem. 274(40):28071-28074 (1999);-   10. Tadokoro et al., Science 302(5642):103-106 (2003);-   11. Moser et al., Nat Med. 14(3):325-330 (2008);-   12. Ginsberg et al., Curr Opin Cell Biol. 17(5):509-516 (2005);-   13. Ma et al., J Thromb Haemost. 5(7):1345-1352 (2007);-   14. Gong et al., Science 327(5963):340-343 (2010);-   15. Wegener et al., Cell 128(1):171-182 (2007);-   16. Goksoy et al., Molecular Cell 31(1):124-133 (2008);-   17. Petrich et al., J Clin Invest 117(8):2250-2259 (2007);-   18. Ren et al., Curr Opin Hematol 15(5):537-541 (2008);-   19. Kataoka et al, Advanced drug delivery reviews 47(1):113-131    (2001);-   20. Dai et al., Blood 106(6):1975-1981 (2005);-   21. Krishnadas et al., Pharm Res. 20(2):297-302 (2003);-   22. Li et al., J Biol Chem 279(41):42469-42475 (2004);-   23. Coller et al., Blood 55:169-178 (1980);-   24. Flevaris et al., J Cell Biol. 179(3):553-565 (2007);-   25. Yin et al., Blood 112(4):1139-1146 (2008);-   26. Yin et al., Blood 111(2):658-665 (2008);-   27. Hayward et al., J Thromb Haemost 4(2):312-319 (2006);-   28. Li et al., Celll 12:77-86 (2003);-   29. Marjanovic et al., J Biol Chem.280(45):37430-37438 (2005);-   30. O'Brien et al., Blood 118(15):4215-4223 (2011);-   31. Day et al., Thromb Haemost 92(3):486-494 (2004);-   32. Falati et al., Nat Med. 8(10):1175-1181 (2002); and-   33. Cho et al., J Clin Invest 118(3):1123-1131 (2008).

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range and each endpoint, unless otherwise indicatedherein, and each separate value and endpoint is incorporated into thespecification as if it were individually recited herein.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein, is intended merely to better illuminate theinvention and does not pose a limitation on the scope of the inventionunless otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element as essential to thepractice of the invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

What is claimed:
 1. A peptide comprising the amino acid sequence Phe Xaa₁ Xaa₂ Glu Xaa₃ Xaa₄ wherein Xaa₁ is Glu and Xaa₂ is Glu or Lys, wherein Xaa₃ is Lys or Arg and Xaa₄ is Met or Leu, wherein the peptide is a 6-mer, 7-mer or 8-mer and is attached to a lipid.
 2. The peptide of claim 1, wherein the lipid is a fatty acid.
 3. The peptide of claim 2, wherein the fatty acid is a C4 to C30 fatty acid, optionally, a C12 to C20 fatty acid.
 4. The peptide of claim 2 wherein the fatty acid is covalently attached to the N-terminus of the peptide.
 5. A micelle comprising (i) the peptide of claim 1, wherein the lipid is optionally a fatty acid, and (ii) at least one second lipid, wherein the second lipid is optionally covalently attached to a water soluble polymer.
 6. The micelle of claim 5, comprising the second lipid covalently attached to a water soluble polymer and a third lipid free of a water soluble polymer, wherein the second lipid, covalently attached to a water soluble polymer is optionally 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000], wherein the third lipid free of a water soluble polymer is optionally phosphatidylcholine.
 7. A pharmaceutical composition comprising the peptide of claim 1, a micelle comprising the peptide of claim 1, or a conjugate comprising the peptide of claim 1, and a pharmaceutically acceptable carrier, diluent, or excipient.
 8. The peptide of claim 1, wherein Xaa3 is Arg.
 9. The peptide of claim 8, wherein Xaa4 is Met.
 10. The peptide of claim 8, wherein Xaa4 is Leu.
 11. The peptide of claim 1, wherein Xaa3 is Lys.
 12. The peptide of claim 11, wherein Xaa4 is Met.
 13. The peptide of claim 11, wherein Xaa4 is Leu.
 14. The peptide of claim 1, wherein the peptide is cyclized.
 15. The peptide of claim 14, comprising two Cys residues, the sulfur atoms of which form a disulfide bridge, optionally, wherein the two Cys residues are terminal residues.
 16. A peptide comprising the amino acid sequence Phe Xaa₁ Xaa₂ Glu Xaa₃ Xaa₄ wherein Xaa₁ is Glu and Xaa₂ is Glu or Lys, wherein Xaa₃ is Lys or Arg and Xaa₄ is Met or Leu, or Ala, wherein the peptide is a 6-mer, 7-mer or 8-mer, wherein the peptide (i) is cyclized, (ii) comprises a fatty acid, optionally, a C4-C30 fatty acid, or (iii) both (i) and (ii).
 17. The peptide of claim 3, wherein the fatty acid is a C14 fatty acid.
 18. A method of inhibiting thrombosis in a subject in need thereof comprising administering to the subject a pharmaceutical composition of claim 7 in an amount effective to inhibit thrombosis.
 19. A method of treating stroke or heart attack in a subject in need thereof comprising administering to the subject a pharmaceutical composition of claim 7 in an amount effective to treat or prevent stroke or heart attack. 